Aus dem Institut für Nutztiergenetik, Mariensee im Friedrich-Loeffler-Institut Experimentelle Untersuchungen zur Transgenese bei Nutztieren Habilitationsschrift zur Erlangung der Venia legendi an der Stiftung Tierärztliche Hochschule Hannover Vorgelegt von Dr. rer. nat. Wilfried August Kues Hannover 2008 1 Für und mit Brigitte 2 Inhaltsverzeichnis 1.0 Eigene Publikationen zur Thematik dieser Habilitationsschrift 4 1.1 Charakterisierung primärer Zellen als Donorzellen für den Kerntransfer 4 1.2 DNA-Mikroinjektion und somatischer Kerntransfer 4 1.3 Übersichtsartikel: Transgene Nutztiere, Stammzellen und DNA-Array Analysen 5 2.0 Einleitung 6 2.1 Die DNA als Träger der genetischen Information und reverse Genetik 6 2.2 Erstellung transgener Nutztiere 8 2.3 Transgene Schweine als Zell- und Organdonoren für die Xenotransplantation 10 2.4 Pharming: Produktion rekombinanter Proteine in der Milchdrüse 12 2.5 Transgene Tiere für landwirtschaftliche Nutzungen 13 2.6 Neuere Methoden der Transgenese 14 2.6.1 Klonen durch somatischen Kerntransfer 14 2.6.2 Gene knock-down in Nutztieren durch RNA-Interferenz 16 2.6.3 Virus-vermittelte Transgenese 16 2.6.4 Konditionale Mutagenese 17 2.6.5 Induzierbare Promotoren und konditionale Expressionskontrolle 18 2.7 Genomprojekte und ihre Bedeutung für die Transgenese 19 2.8 Epigenetik 22 3.0 Fragestellungen 24 4.0 Ergebnisse und Diskussion 25 4.1 Primäre somatische und Stamm-Zellen als Donorzellen für den Kerntransfer 25 4.2 Gene knock-down-Versuche in transgenen Embryonen 29 4.3 Generierung transgener Schweine mit konditionaler Transgenregulation 32 5.0 Zusammenfassung 39 6.0 Literaturverzeichnis 40 7.0 Danksagungen 54 8.0 Darstellung des eigenen Anteils an den wissenschaftlichen Arbeiten 55 9.0 Abkürzungsverzeichnis 59 10.0 Anhang: Nachdruck der eigenen Publikationen 60 3 1.0 Eigene Publikationen zur Thematik dieser Habilitationsschrift 1.1 Charakterisierung primärer Zellen als Donorzellen für den Kerntransfer Anger, M., Kues, W.A., Klima, J., Mielenz, M., Motlik, J., Carnwath, J.W. and Niemann, H. 2003. Cloning and cell cycle-specific regulation of Polo-like kinase in porcine fetal fibroblasts. Molecular Reproduction and Development 65, 245-253. Kues, W. A., Petersen, B., Mysegades, W., Carnwath, J.W. and Niemann, H. 2005. Isolation of fetal stem cells from murine and porcine somatic tissue. Biology of Reproduction 72, 1020-1028 Yadav, P., Kues, W.A., Herrmann, D., Carnwath, J.W., Niemann, H. 2005. Bovine ICMs express the Oct4 ortholog. Molecular Reproduction and Development 72, 182-190. Dieckhoff, B., Karlas, A., Hofmann, A., Kues, W.A., Petersen, B., Pfeifer, A., Niemann, H., Kurth, R., Denner, J. 2007. Inhibition of porcine endogenous retroviruses (PERVs) in primary porcine cells by RNA interference using lentiviral vectors. Archives of Virology 152, 629-34. 1.2 DNA-Mikroinjektion und somatischer Kerntransfer Schätzlein S., Lucas-Hahn, A., Lemme, E., Kues, W.A., Dorsch, M., Manns, M., Niemann, H., Rudolph, K.H. 2004. Telomere length is reset during early embryogenesis. Proceedings of the National Academy of Sciences of the USA 101, 8034-8038. Hölker, M., Petersen, B., Hassel, P., Kues, W.A., Lemme, E., Lucas-Hahn, A., Niemann, H. 2005. Duration of in vitro maturation of recipient oocytes affects blastocyst development of cloned porcine embryos. Cloning and Stem Cells 7, 34-43. Kues, W.A., Schwinzer, R., Wirth, D., Verhoeyen, E., Lemme, E., Herrmann, D., Barg-Kues, B., Hauser, H., Wonigeit, K., Niemann H. 2006. Epigenetic silencing and tissue independent expression of a novel tetracycline inducible system in double-transgenic pigs. FASEB Journal 20, E1-E10, doi: 10.1096/fj.05-5415fje. Deppenmeier, S., Bock, O., Mengel, M., Niemann, H., Kues, W., Lemme, E., Wirth, D, Wonigeit, K., Kreipe, H. 2006. Health status of transgenic pig lines expressing human complement regulator protein CD59. Xenotransplantation 13, 345-356. Hornen, N., Kues, W.A., Carnwath, J.W., Lucas-Hahn, A., Petersen, B., Hassel, P., Niemann, H. 2007. Production of viable pigs from fetal somatic stem cells. Cloning and Stem Cells 9, 364-73. Iqbal, K, Kues, W.A., Niemann, H. 2007. Parent of origin dependent gene-specific knock down in mouse embryos. Biochemical and Biophysical Research Communications 358, 727-732. 4 Dieckhoff, B., Petersen, B., Kues, W.A., Kurth, R., Niemann, H., Denner, J. 2008. Knockdown of porcine endogenous retrovirus (PERV) expression by PERV-specific shRNA in transgenic pigs. Xenotransplantation 15, 36-45. 1.3 Übersichtsartikel: Transgene Nutztiere, Stammzellen und DNA-Array Analysen Niemann, H., und Kues, W.A. 2003. Application of transgenesis in livestock for agriculture and biomedicine. Animal Reproduction Science 79, 291-317. Kues, W.A. und Niemann, H. 2004. The contribution of farm animals to human health. Trends in Biotechnology 22, 286-294. Kues, W.A., Carnwath, J.W., Niemann, H. 2005. From fibroblasts and stem cells: Implications for cell therapies and somatic cloning. Reproduction, Fertility and Development 17, 125-134. Niemann H, Kues W, Carnwath JW. 2005. Transgenic farm animals: present and future. OIE Scientific and Technical Reviews (Rev. Sci. Tech. Off. Int. Epiz.) 24, 284-298. Niemann, H. und Kues, W.A. 2007. Transgenic farm animals – an update. Reproduction, Fertility and Development 19, 762-770. Niemann, H., Carnwath, J.W., Kues, W.A. 2007. Application of array technology to mammalian embryos. Theriogenology 68S, S165-S177. Kues, W.A., Rath, D., Niemann, H. 2008. Reproductive biotechnology goes genomics. CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources 3, No. 036, 1-18. 5 2.0 Einleitung 2.1 DNA als Träger der genetischen Information und reverse Genetik Die Desoxyribonucleinsäure (DNS, bzw. DNA) ist der Träger der genetischen Information. DNA wurde 1869 erstmals von Friedrich Miescher aus Eiterzellen isoliert, ihre biologische Funktion als Informationsträger wurde aber erst 1944 durch Transformation von Pneumokokken gezeigt (Avery et al., 1944). Die Aufklärung der DNA-Molekülstruktur als Doppelhelix (Watson und Crick, 1953) erlaubte erstmals einen Einblick, wie biologische Information gespeichert, repliziert und abgelesen wird und bildete eine wichtige Grundlage für die Entwicklung der Molekularbiologie. Seit ca. 1970 wurde es möglich, in kurzen DNA-Stücken die Basenabfolge zu bestimmen (DNA-Sequenzierung), sowie DNA-Stücke gezielt neu zusammen zu setzen und die Eigenschaften der rekombinanten DNA in vitro und in verschiedenen einfachen Modellorganismen, vorwiegend Escherichia coli (E. coli), oder in eukaryotischen Zell-Linien, zu testen (Sanger et al., 1973; Maxam und Gilbert, 1977; Sanger et al., 1977; Jackson et al., 1972; Johnson, 1983; Watson et al., 1983, Mulligan et al., 1979). So konnten grundlegende molekulare Eigenschaften der DNA wie der Aufbau von Genen, regulatorischen Sequenzen (Promotor, Enhancer, Silencer) und die Intron-Exon-Organisation eukaryotischer Gene aufgeklärt werden (Glover, 1985; Sambrook et al., 1989). Ein Standardverfahren in E.coli ist die Transformation mit ringförmigen DNA-Molekülen (Plasmide), die eine AntibiotikaResistenz und einen Startpunkt für die Replikation (origin of replication) in ihrer Sequenz tragen. Zusätzlich kann Fremd-DNA in ein Plasmid ligiert werden, typischerweise 1 000 – 20 000 Basenpaare (1-20 kBp). Die Plasmide liegen episomal in E. coli vor und werden bei jeder Zellteilung repliziert (Glover, 1985; Sambrook et al., 1989). Falls die Fremd-DNA prokaryotische Promotorelemente und eine protein-kodierende Sequenz enthält, wird diese Information abgelesen und das rekombinante Protein exprimiert. Zahlreiche Medikamente werden mittlerweile rekombinant in Bakterien hergestellt. Humanes Insulin, Wachtumshormon, Erythropoietin und Interferon sind die umsatzstärksten Medikamente (Blockbuster) mit jeweils mehr als 1 Milliarde $ Umsatz pro Jahr (Nightingale und Martin, 2004). In Prokaryoten erfolgt im wesentlichen eine direkte Ablesung der genetischen Information, im Eukaryoten liegt eine wesentlich komplexere Regulation der Genaktivität vor, die bisher erst teilweise entschlüsselt ist. 6 Bereits 1971 wurden durch Inkubation von Spermien mit Fremd-DNA transgene Embryonen erstellt (Brackett et al, 1971), bzw. durch Infektion präimplantatorischer Maus-Embryonen mit Simian Virus 40 (Jaenisch und Mintz, 1974) oder Moloney Leukemia-Viren (Jaenisch, 1976) transgene Mäuse erzeugt, die die Fremd-DNA in ihr Genom integriert hatten, und an ihre Nachkommen weitergaben. Trotz Veränderung des Genotyps (reverse genetics) dieser Tiere, wurden keine phänotypischen Veränderungen gezeigt. Der erste Nachweis, dass eine rekombinante DNA-Sequenz eine phänotypische Änderung im Säuger zur Folge hatte, wurde 1982 publiziert (Palmiter et al. 1982, Abb.1). Diese Arbeit ist ein Meilenstein in der Genetik und die Grundlage für ein vertieftes molekulares Verständnis der Gen-Phänotyp-Beziehung und für transgene Forschungsansätze, die darauf abzielen, die komplexe Genregulation in Eukaryoten und speziell in Säugern zu verstehen. In dieser Arbeit wurde ein Expressionskonstrukt mit dem Metallothionein-Promoter und der cDNA des Rattenwachstumshormons (rGH) verwendet. Das aufgereinigte DNA-Expressionskonstrukt wurde dann direkt in den männlichen Vorkern von Mauszygoten injiziert, die anschließend in den Eileiter von scheinschwangeren Empfängertieren übertragen wurden. Abb. 1. Verstärktes Wachstum in transgenen Mäusen. Transgene Maus (links), in der die Expression des Ratten-Wachstumshormongens zu verstärktem Wachstum führt; zum Vergleich ein nicht-transgenes Geschwistertier (rechts) (Palmiter et al., 1982). Die Integration der rGH-Sequenz in das Mausgenom wurde in den Nachkommen durch Southern blot-Nachweis mit einer markierten komplementären Sonde gezeigt. Die transgenen 7 Tiere zeigten phänotypisch ein verstärktes Wachstum, das durch die erhöhten Serumspiegel des Wachstumshormons hervorgerufen wurde. Transgene Mausmodelle sind mittlerweile ein Standardwerkzeug der molekularbiologischen Forschung geworden, sie werden für das Verständnis genetischer Erkrankungen, wie Parkinson, Alzheimer, Trisomie 21, sowie die Entwicklung von Therapiemöglichkeiten in der Immunologie, der Krebsforschung und der Grundlagenforschung verwendet (Dodart und May, 2005; Yang und Gong, 2005; Scharschmidt und Segre, 2008). Insbesondere die Möglichkeit über murine embryonale Stammzellen (ES) gezielt spezifische Gene auszuschalten (Gene knock-out) hat die Anwendungsfelder der reversen Genetik vergrößert (Mansour et al., 1988; Capecchi, 1989; Götz et al., 1998; Müller, 1999). Weitere Verfeinerungen des Methodenrepertoires sind die konditionale Expression von Transgenen über Rekombinasen (z.B. Cre/loxP-System), die konditionale Expression von binären Expressionskassetten (tet-on, tet-off) oder die RNA-Interferenz (RNAi) mittels kurzer doppelsträngiger RNAs (siRNA oder shRNA) (Orban et al., 1992; Gu et al., 1994; Furth et al., 1994; Zhou et al., 2006). Allerdings hat das Mausmodell neben zahlreichen Vorteilen auch Beschränkungen, die sich insbesondere aus den gravierenden anatomischen und physiologischen Unterschieden zwischen Maus und Mensch ergeben (Habermann et al., 2007; Wall und Shani, 2008; Taft, 2008). Daher ist das Mausmodell für spezifische Fragestellungen, z.B. in Transplantationsmedizin, Kreislauf/Bluthochdruckforschung oder Gerontologie aufgrund der geringen Größe und kurzen Lebensdauer nur sehr bedingt aussagekräftig. 2.2 Erstellung transgener Nutztiere Transgene Nutztiere stellen für spezifische biomedizinische Fragestellungen eine interessante Alternative zum Mausmodell dar. Hierzu zählen insbesondere Xenotransplantation, Pharming (Produktion rekombinanter Proteine in der Milchdrüse), Bluthochdruck- und Altersforschung. Auch in der Landwirtschaft kann eine gezielte genetische Veränderung von Nutztieren wesentliche Vorteile für eine effizientere und ressourcenschonendere Produktion haben (Wall, 1996). Die ersten genetisch veränderten Schweine, Schafe und Kaninchen wurden 1985 durch DNAMikroinjektion in Zygotenstadien erstellt (Hammer et al., 1985, Brem et al., 1985). In der Folgezeit wurde über transgene Ansätze versucht, verschiedene Eigenschaften von Nutztieren zu verbessern: Wachstum und Futterverwertung (Pursel, 1998; Golovan et al., 2001), Milchleistung (Zuelke, 1998; Brophy et al., 2003), Wolleigenschaften (Damak et al., 1996), 8 sowie Reproduktion und Krankheitsresistenz (Müller et al., 1992; Clements et al., 1994; Müller und Brem, 1994; Wolf et al., 2000). Als wesentlicher Nachteil dieses Verfahrens erwies sich, dass die Erfolgsrate transgener Nachkommen mit durchschnittlich 5-10% der geborenen Tiere sehr gering ist (Abb.2). Bezogen auf die mikroinjizierten Embryonen liegen die Erfolgsraten bei 1-3% (Abb.2). Im Gegensatz zur Maus, weisen Zygoten von Rind und Schwein einen sehr hohen Anteil von Lipiden auf, die die DNA-Mikroinjektion in Zygotenvorkerne erschweren. Nur durch zusätzliche Schritte, wie Zentrifugation der Zygoten, können die Vorkerne dargestellt werden (Abb.3). Erschwerend kommt hinzu, dass von den transgenen Trägertieren (founder animals) nur ein Teil das Transgen vererbt (Keimbahntransmission) und die gewünschte Expression aufweist (Hogan et al., 1996). Da die Fremd-DNA zufallsmäßig in das Genom integriert, können Mosaik-Integration, insertionale Mutationen, variable Kopienzahlen des Transgens und variable Expression durch Positionseffekte benachbarter DNA-Sequenzen auftreten (Hogan et al., 1986; Deppenmeier et al., 2006). Maus Kaninchen Schwein Schaf Rind 0 5 10 15 [%] Abb. 2. Effizienz der DNA-Vorkerninjektion in verschiedenen Säugerspezies. Die Transgenese-Effizienz ist als relativer Anteil der transgenen Tieren an den geborenen Nachkommen (gestrichelte Säulen) und an den DNA-injizierten und auf Empfängertier übertragenen Embryonen (schwarze, gefüllte Säulen) angegeben (verändert nach Wall, 1996). In den letzten Jahren wurde deutlich, dass an der komplexen Regulation der Genomaktivität in Eukaryoten auch zahlreiche sogenannte epigenetische Mechanismen (Epi-Genetik = „Über“-Genetik) beteiligt sind (Reik, 2007). Als Epigenetik bezeichnet man vererbbare Eigenschaften, die nicht direkt in der DNA-Sequenz verankert sind. Relativ gut untersucht sind Methylierungen der Cytosin-Base in CG-Dinukleotiden (CpG) bei Säugern, sowie 9 Methylierungen, Azetylierungen und Phosphorylierungen der Histon-Proteine (Chandler und Jones, 1988; Bird, 1992; Li, 2002; Maatouk et al., 2006). Bedingt durch die hohen Kosten für die Erstellung transgener Nutztiere, die erforderliche umfangreiche Logistik und die langen Reproduktionszeiten, fokussierte sich die Forschung im wesentlichen auf biomedizinisch relevante Gebiete wie Xenotransplantation und Pharming. Insbesondere bei Nutztieren, aber auch in der Maus sind die Probleme der Transgenese mit häufig ungenügender Expression, oder ektopischer Expression nur teilweise verstanden. Gegenwärtig ist es aufgrund der unzureichenden Vorhersagbarkeit der transgenen Expression, immer noch notwendig, zahlreiche Linien auf die gewünschten Expressionseigenschaften zu untersuchen. Bei der Maus stellt das aufgrund der kurzen Generationszeiten und der relativ geringen Kosten kein grundlegendes Problem dar, bei Nutztieren ergeben sich enorme Kosten bei der Erstellung und Charakterisierung. Es wird geschätzt, dass die Kosten für die Erstellung eines transgenen Rindes ca. 100 000 $ betragen, bei Schweinen werden Kosten von ca. 25 000 $ genannt (Wall, 1996). A B Promotor Transgen-cDNA Poly(A) Abb. 3. DNA-Mikroinjektion in einen Vorkern einer porcinen Zygote. A) Nach Zentrifugation bei 10 000 g ist die undurchsichtige Lipidfraktion des Zytoplasmas in einer Zellhälfte konzentriert. In einen der dadurch sichtbar gewordenen Vorkerne wird mit Hilfe einer Mikrokapillare (rechte Bildhälfte) die DNA-Lösung injiziert. B) Schematische Darstellung eines Minigen-DNA-Konstrukts bestehend aus einem Promotor, einer protein-kodierenden cDNA und einer Polyadenylierungssequenz, die die Stabilität der abgelesenen mRNA erhöht. Daraus ergibt sich, dass für einen breiteren Einsatz der Transgenese bei Nutztieren, die Effizienz der Transgenese erhöht und die Kosten deutlich sinken müssen. Trotz dieser Schwierigkeiten sind in den letzten Jahren wesentliche Fortschritte in der Erstellung transgener Nutztiere erzielt worden. 2.3 Transgene Schweine als Zell- und Organdonoren für die Xenotransplantation Die Erfolge der Allotransplantation, d.h. die Transplantation humaner Organe in Patienten mit Organausfall, hat zu einer dramatischen Verknappung der verfügbaren Organe geführt. Selbst 10 eine verbesserte Allokation und die Lebendspende von Organen können den Bedarf bei weitem nicht decken (Deutsche Stiftung Organtransplantation: www.dso.de). In Deutschland stehen ca. 12 000 Patienten auf der Warteliste für eine Nierentransplantation, pro Jahr sind aber nur knapp 4 000 Spendernieren verfügbar. Für andere Organe, wie Herz, Leber und Lunge können mit den verfügbaren Spenderorganen jeweils nur ein Drittel bis die Hälfte der notwendigen Transplantationen durchgeführt werden. Aufgrund der Organmangelsituation versterben in Deutschland pro Tag im Durchschnitt drei Patienten auf der Warteliste an akutem Organversagen. Eine mögliche Alternative stellt die Transplantation von Tierorganen (Xenotransplantation) dar (White, 1996; Bach, 1998, Niemann und Kues, 2000). Wesentliche Voraussetzungen für klinische Studien mit Xenotransplantaten sind, dass die immunologischen Abstoßungsreaktionen vermindert oder überwunden werden können, die Übertragung von Tierpathogenen ausgeschlossen werden kann, und eine weitgehende physiologische Kompatibilität zu dem entsprechenden Humanorgan besteht. Das Schwein stellt dabei die am besten geeignete Spezies dar, da die technischen Möglichkeiten zur genetischen Modifikation des Schweinegenoms vorhanden sind, die Haltung unter kontrollierten Bedingungen in spezifisch pathogenfreien (SPF) oder gnotobiotischen Anlagen etabliert ist, aufgrund der multiparen Reproduktion sehr schnell zahlreiche Nachkommen erzeugt werden können und eine grosse Ähnlichkeit in der Physiologie und Anatomie zum Menschen bestehen (Niemann und Kues, 2000). Bisher sind im wesentlichen transgene Schweine mit Expression humaner Komplementregulatoren (regulators of complement activation – RCA) (McCurry et al., 1995; Cozzi et al., 1997; Zaidi et al., 1998; Niemann et al., 2001), bzw. mit einem Gene knock-out der α-1,3-Galactosyltransferase (Dai et al. 2002; Lai et al., 2002) für Xenotransplantationsmodelle produziert worden. Beide methodischen Ansätze setzen auf molekularer Ebene auf der Unterdrückung der hyperakut ablaufenden Komplementvermittelten Abstoßung an (Yang-Guang und Sykes 2007; Zhong, 2007). Nach Xenotransplantation von RCA-transgenen, bzw Gal-knock-out Nieren und Herzen in nichthumane Primaten wurden maximale Überlebenszeit von 179 Tagen erreicht, die medianen Überlebenszeiten liegen aber deutlich unter 60 Tagen (McGregor et al., 2004; Kuwaki et al., 2005; Yamada et al., 2005). Obwohl die geringen Transplantationszahlen keine statistisch signifikanten Aussagen erlauben, wird angenommen, dass zu den bisherigen genetischen Modifikationen weitere Transgene in das Schweinegenome eingeführt werden müssen, um die nachfolgenden Abstoßungsreaktionen, wie die akut vaskuläre Abstoßung (AVR) und die 11 zelluläre Abstoßung kontrollieren können und dadurch längere Überlebenszeiten zu ermöglichen. 2.4 Pharming: Produktion rekombinanter Proteine in der Milchdrüse 2006 wurde von der European Medicine Agency (EMEA) erstmalig ein rekombinantes Protein aus der Milchdrüse als Medikament zugelassen (Niemann und Kues, 2007). Rekombinantes humanes Antithrombin III wird in der Milch transgener Ziegen produziert und ist zunächst für die prophylaktische Behandlung von Patienten mit Antithrombindefizienz vorgesehen. Inzwischen ist die Entwicklung fortgeschritten und zahlreiche weitere rekombinante Proteine aus der Milchdrüse von Kaninchen, Ziegen, Schafen oder Kühen befinden sich in der klinischen Prüfung (Tab.1). Tab. 1: Entwicklungsstand in der Produktion rekombinanter Proteine aus der Milchdrüse von Nutztieren Rekombinantes Protein Hersteller Tierart Klinische Prüfung humanes Antithrombin III GTC Biotherapeutics Ziege EU: zugelassen (Produktname: ATryn) USA: Phase 3 C1 Estera se Inhibitor Pharming Kaninchen Phase 3 MM-093 (AFp) Merrimack und GTC Ziege Phase 2 α-Glucosidase Pharming Kaninchen Phase 2 (gestoppt) humanes Wach stumshormon BioSidus Rind Vorklinisch Albumin GTC Biotherapeutics Rind Vorklinisch Fibrinogen Pharming Rind Vorklinisch Collagen Pharming Rind Vorklinisch Lactoferrin Pharming Rind Vorklinisch α-1 Antitrypsin GTC Biotherapeutics Ziege Vorklinisch Malaria-Vakzin GTC Biotherapeutics Ziege Vorklinisch CD137 monoklonaler AK GTC Biotherapeutics Ziege Vorklinisch (verändert nach Echelard et al., 2006) Die Milchdrüse weist für die rekombinante Proteinproduktion eine Reihe von Vorteilen auf. So können Prokaryoten keine posttranslatorischen Glykosylierungen durchführen, die für die Aktivität zahlreicher eukaryotischer Proteine notwendig sind. Die Produktion komplexer Proteine in eukaryotischen Zellkulturen ist häufig nur mit geringer Syntheseleistung und Ausbeute möglich und entsprechend kostenaufwendig. Die Milchdrüse ist für hohe metabolische Syntheseraten von Proteinen ausgelegt, Milch kann durch Melken einfach gewonnen und für die Aufreinigung der rekombinanten Proteine verwendet werden (Rudolph, 1999; Echelard et al., 2006). 12 2.5 Transgene Tiere für landwirtschaftliche Nutzungen Tabelle 2 zeigt eine Zusammenfassung der wichtigsten transgenen Ansätze zur Verbesserung von Nutztieren für die landwirtschaftliche Produktion. So sind transgene Schweine erstellt worden, die ein Desaturase-Gen im Fettgewebe produzieren und dadurch ein höheres Verhältnis von vielfach ungesättigten zu gesättigten Fettsäuren im Muskelfleisch aufweisen (Saeki et al., 2004; Lai et al., 2006). Insbesondere Omega-3-Fettsäuren wurden vermehrt gebildet. Eine Diät mit hohem Anteil ungesättigter Fettsäuren wird mit einem verminderten Risiko für Herzkreislauferkrankungen korreliert, und daher von den Gesundheitsbehörden empfohlen. Die Expression von bovinem Lactalbumin in der Milchdrüse von Schweinen führte zu einem höheren Laktose-Gehalt und erhöhter Milchproduktion, was wiederum zu besseren Entwicklungsraten der neugeborenen Ferkel führte (Wheeler et al., 2001). Durch die Expression bakterieller Phytase in der Speicheldrüse von Schweinen können diese Tiere sonst unverdaubares pflanzliches Phytat metabolisieren (Golovan et al., 2001). Dadurch wird die Phosphat-Belastung in der Gülle signifikant vermindert. Tab. 2 Transgene Nutztiere für landwirtschaftlich wichtige Merkmale Tierart Transgen/Konstrukt Phänotyp Methode Referenz Schwein Wachstumshormon/hMT-pGH verbessertes Wachstum, weniger Körperfett Mikroinjektion Nottle et al., 1999 Schwein Insulin-like Wachstumsfaktor verbessertes Wachstum Mikroinjektion Pursel et al., 1999 Saeki et al., 2004 Schwein Desaturase/maP2-FAD erhöhte Werte für mehrfach ungesättigte Fettsäuren Mikroinjektion Schwein Desaturase/CAGGS-hfat-1 erhöhte Werte für mehrfach ungesättigte Fettsäuren somatischer Kerntransfer Lai et al., 2006 Schwein Phytase/PSP-APPA Phytat-Metabolisierung Schwein Laktalbumin/genomische DNA erhöhter Laktosegehalt der Milch Schwein Mx-Protein/mMx1-Mx Mikroinjektion Golovan et al., 2001 Mikroinjektion Wheeler et al. 2001 Influenza-Resistenz Mikroinjektion Müller et al., 1992 Schwein/Schaf IgA/a,K-a,K Pathogen-Resistenz Mikroinjektion Lo et al., 199 Schaf Insulin-like Wachstumsfaktor Wollwachstum Mikroinjektion Damak et al., 1996 Schaf PRNP knock-out-Konstrukt Scrapie-Resistenz (Tiere neonatal verstorben) somatischer Kerntransfer Denning et al. 2001 Ziege Stearoyl-CoA Desaturase veränderte Milchzusammensetzung Mikroinjektion Rind Kaseine/genomische DNA veränderte Milchzusammensetzung somatischer Kerntransfer Brophy et al., 2003 Rind Lysostaphin Staphylococcus aureus-Resistenz somatischer Kerntransfer Wall et al., 2005 Reh et al., 2004 (verändert aus Niemann und Kues, 2007) Der Einsatz dieser Tiere in der kommerziellen Fleischproduktion würde den kostspieligen Futterzusatz von rekombinanten Phytasen oder organischem Phosphor vermeiden und einen wesentlichen Beitrag zu verminderten Phosphat-Emissionen liefern. Über Gene knock-out und Gene knock-down-Techniken wurden Rinder mit einer Deletion des Prionprotein-Gens (PRNP), bzw. mit signifikant verminderten PRNP-mRNA-Werten erstellt (Kuroiwa et al., 2004; Golding et al., 2006; Richt et al., 2007). Diese sollten genetisch 13 resistent gegen die bovine spongiforme Enzephalitis (BSE) sein, erste Ergebnisse aus in vitro Versuchen scheinen diese Resistenz zu bestätigen (Richt et al., 2007). BSE-resistente Rinder sind insbesondere für das Pharming von Interesse, da hier alle Produktionsschritte nach Good Manufacturing Practices (GMP) durchgeführt werden müssen, und Freiheit von möglichen Pathogenen gewährleistet sein muss. Bisher sind keine transgenen Tiere für die landwirtschaftliche Produktion zugelassen. Mittlerweile werden transgene Zierfische kommerziell in den USA, Hong Kong, Taiwan und Malaysia gehandelt (Niemann und Kues, 2007). Bei den Medaka (Oryzias latipes) und Zebrabärblingen (Danio rerio) werden verschiedene fluoreszente Proteine im Muskelgewebe exprimiert (Chou et al., 2003; Gong et al., 2003), die die Eigenfärbung der Fische überstrahlen. Ein Bericht der Food and Drug Administration der USA (FDA, 2003) stellte fest, dass diese transgenen Fische unbedenklich sind. Die transgenen Zierfische können möglicherweise zu einer veränderten Einstellung der Öffenlichkeit gegenüber gentechnisch veränderten Tieren beitragen. 2.6 Neuere Methoden der Transgenese 2.6.1 Klonen durch somatischen Kerntransfer 1997 wurde erstmals das erfolgreiche Klonen eines Säugers über den Kerntransfer einer somatischen Zelle in eine entkernte Oozyte beschrieben (Wilmut et al., 1997). Damit wurde gezeigt, dass somatische Zellen grundsätzlich reprogrammierbar sind und nach Reprogrammierung eine komplette Ontogenese durchlaufen können. In der Folge wurde das somatische Klonen zu einer Schlüsseltechnik für die Grundlagenforschung, um Prozesse der Reprogrammierung, der Pluripotenz und der zellulären Differenzierung untersuchen zu können. Daneben wird das Klonen von genetisch wertvollen Tieren durch somatischen Kerntransfer mittlerweile kommerziell angeboten (Panarace et al., 2006). Ein weiterer wichtiger Aspekt ist, dass über das Klonen auch transgene Ansätze ermöglicht wurden, die zuvor im Nutztier nicht verfügbar waren (Cibelli et al., 1998). So können in der Maus über etablierte ES-Zellen Gene knock-out-Mutanten erhalten werden. Für Nutztiere sind bis heute keine echten ES-Zellen isoliert worden, und die klassische Knock-out-Technik ist daher nicht verfügbar. Mit dem somatischen Klonen eröffnet sich die Möglichkeit, zunächst einen Gene knock-out in primären somatischen Zellen durchzuführen (Schnieke et al., 1997; Dai et al., 2002; Lai et al., 2002), und vorselektionierte Zellen mit Knock-out dann als Kernspenderzellen einzusetzen (Kolber-Simonds et al., 2004; Abb. 4). 14 Über die homologe Rekombination in primären Fibroblasten wurden Schafe mit einem Genknock-out für das Prion Protein (PRNP) geklont, die allerdings kurz nach der Geburt verstarben (Denning et al., 2001). Im Rind konnten vitale Tiere mit PRNP-knock-out erhalten werden (Kuroiwa et al., 2004; Richt et al., 2007). Im Schwein wurden Knock-out Tiere für die α-1,3- Galaktosyltransferase erstellt (Dai et al., 2002; Lai et al., 2002), die in der Xenotransplantation eine wesentliche Rolle spielt (siehe 2.3). Targeting-Konstrukt Isolation rekombinierter Zellklone entkernte Oozyte Elektroporation homologe Rekombination Somatischer Kerntransfer Zweite Durchmusterung auf ein „loss of heterozygosity“Ereignis Embryotransfer auf synchronisiertes Empfängertier -/Gen knock-out Tier Abb.4 Erstellung von Gene knock-out Schweinen durch somatischen Kerntransfer (mod. aus Niemann u. Kues, 2003) Über einen sogenannten Gene knock-in wurde der Blutgerinnungsfaktor IX gezielt in einen aktiven Genbereich des ovinen Genoms integriert und anschließend über den somatischen Kerntransfer vitale Schafe geklont (Schnieke et al., 1997). Das somatische Klonen hat entscheidende Vorteile für die Transgenese von Nutztieren. Allerdings sind neben dem hohen mikromanipulatorischen Aufwands für den Kerntransfer (Du et al., 2005) auch neue Probleme (Young et al., 1998; DeSousa et al., 2001) und Kosten entstanden. So war die ursprüngliche Ansicht, dass nur Zellen in der G0 oder G1-Phase des Zellzyklus als Kernspender geeignet seien (Wilmut et al., 1997). Dies scheint aber aus heutiger Sicht für den Klonerfolg nicht entscheidend zu sein. Auch die Frage, ob es 15 Unterschiede verschiedener somatischer Zellen in Bezug auf Differenzierungsgrad, Ausgangsgewebe, oder Passagenzahl in vitro gibt, ist nicht abschließend geklärt (Kues et al., 2000; Kues et al., 2005b). Bei der Maus konnte gezeigt werden, dass auch terminal ausdifferenzierte Zellen aus dem Nerven- und Blutsystem zu Klonnachkommen führen können (Hochedlinger und Jaenisch, 2002; Eggan et al., 2004), allerdings mit stark reduzierter Effizienz verglichen zu Fibroblasten. Porcine fetale Stammzellen und Fibroblasten zeigten ähnliche Kloneffizienzen (Hornen et al., 2007). Fibroblasten dienen in den meisten Klonversuchen als Kerndonoren. Fibroblasten sind relativ leicht zu kultivieren und benötigen keine Supplementation mit spezifischen Wachstumsfaktoren in der in vitro Kultur. Allerdings handelt es sich hier stets um heterogene Zellpopulationen mit beschränkter Proliferation in vitro (Hayflick, 1965). 2.6.2 Knock-down in Nutztieren durch RNA interference Eine Alternative zum klassischen Gene knock-out ist der Gene knock-down, bei dem über RNA-Interferenz eine Degradation von spezifischen mRNAs erreicht wird (Fire et al., 1998; Jorgensen et al., 1998; Sharp, 2001). Bei Säugern werden dabei kurze doppelsträngige RNAs (17-25 Bp), sogenannte short interfering RNA (siRNA), bzw. short hairpin RNA (shRNA), vom RNA-Induced Silencing Complex (RISC) gebunden, und mRNAs mit komplementären Sequenzbereichen selektiv degradiert (Elbashir et al., 2001). Man vermutet, dass der RNAiMechnismus neben der Genregulation vor allem eine Funktion in der Virusresistenz hat. Durch das zelluläre Dicer-Enzym werden doppelsträngige RNAs, wie sie in RNA-Viren als Genom vorkommen, erkannt und zu siRNAs prozessiert. Diese „maßgeschneiderten“ virusspezifischen siRNAs führen dann zu einer Degradation von viralen mRNAs. Für die Transgenese können siRNA-Expressionskonstrukte in das Genom eingebracht werden und den funktionellen Verlust eines spezifischen Transkriptes bewirken. Da keine Veränderung auf der Ziel-DNA-Sequenz notwendig ist, ist eine Zucht auf Homozygotie, wie bei Gene knock-out Ansätzen nicht notwendig (Tenenhaus-Dann et al., 2006). In Kombination mit neuartigen Vektoren für die Transgenese, wie Lentiviren, ist damit im Prinzip eine direkte genetische Veränderung von Produktionslinien von Nutztieren möglich. 2.6.3 Virus-vermittelte Transgenese Die ersten viralen Ansätze zur Transgenese bei Mäusen mit SV40 oder Moloney LeukemiaVirus (Jaenisch und Mintz, 1974; Jaenisch, 1976) und mit Retroviren bei Rindern (Haskell and Bowen, 1995; Chan et al., 1998) führten zu Tieren, die die Fremd-DNA im Genom 16 integriert trugen; die Fremd-DNA war jedoch stillgelegt (DNA silencing) und die Tiere damit phänotypisch unverändert. In den letzten Jahren zeigte sich, dass Lentiviren, eine Unterklasse der Retroviren, deutlich bessere Vektoren für die Erstellung transgener Mäuse und Nutztiere darstellen (Pfeifer et al., 2002; Hofmann et al., 2003 u. 2004; Whitelaw et al., 2004). So können transgene Tiere direkt durch Injektion der Lentiviren in den perivitellinen Raum von Zygotenstadien erzeugt werden. Dabei wurde eine sehr hohe Rate transgener Nachkommen beobachtet, in denen eine aktive Expression der Transgene festgestellt wurde. Offenbar kommt es durch die veränderte Zusammensetzung der terminalen Repeat-Bereiche bei den Lentiviren nicht zu einem DNA-Silencing im Säugergenom. Ein weiterer Faktor ist, dass nach lentiviraler Infektion multiple Integrationen auf verschiedenen Chromosomen stattfinden. Inwiefern dies zu einer höheren Rate von Integrationsmutationen führt, muss noch abgeklärt werden. 2.6.4 Konditionale Mutagenese Eine weitere Verfeinerung des Methodenrepertoires stellt die konditionale Mutagenese dar. So kann ein konventioneller Gene knock-out zunächst einmal nicht räumlich oder zeitlich beschränkt werden. Für viele Untersuchungen ist es aber wünschenswert, ein Gen zu einem definierten Zeitpunk aus- oder anzuschalten, um z.B. kompensatorische Mechanismen, die sich bei einem permanenten Knock-out entwickeln können, zu vermeiden. Auch können so Gene, die essentiell für die Embryogenese sind und Letalmutationen darstellen, gezielt nach der Geburt ausgeschaltet werden. Die Methoden für die konditionale Mutagenese sind im wesentlichen in der Maus entwickelt worden. So ist die Cre-spezifische DNA-Rekombinase des Bakteriophagen P1 auch in Säugerzellen aktiv und kann spezifische Rekombinationen von DNA-Sequenzen durchführen, die von als „loxP“ bezeichneten Erkennungssequenzen flankiert sind (Sauer, 1998). Das 38kD Cre Protein benötigt dabei keine weiteren Cofaktoren. Je nach Orientierung der loxP-Erkennungssequenzen zueinander, kommt es dabei zur Exzision der flankierten DNA-Region oder zur Inversion (Sauer und Henderson, 1988). Durch das Einführen der loxP-Erkennungssequenzen in ein Zielgen und die zusätzliche Präsenz des Cre-Gens unter der Kontrolle eines gewebespezifischen Promoters kann ein beschränkter gewebespezifischer Knock-out erreicht werden (Orban et al., 1992; Gu et al., 1994; St-Onge et al., 1996). Für diesen Ansatz sind zwei Linien transgener Mäuse notwendig. Die erste Linie trägt dabei die Cre-Rekombinase unter der Kontrolle eines Promoters mit dem gewünschten Expressionsprofil. In der zweiten Linie wird die DNA-Zielregion über homologe Rekombination in ES-Zellen mit loxP-Sequenzen flankiert. Durch die loxP17 Sequenzen wird die normale Expression nicht beeinträchtigt. Durch Linien-Verpaarung werden beide Modifikationen in einem Genom kombiniert. So zeigen DNA-Polymerase ßdefiziente Mäuse einen embryonal-lethalen Phenotyp. Durch konditionale Mutagenese war es möglich, lebensfähige Nachkommen mit loxP-flankiertem DNA-Polymerase ß-Gen zu erhalten und selektiv einen Knock-out in T-Zellen zu generieren (Gu et al., 1994). Eine wichtige Voraussetzung, um mögliche embryonal-letalen Phenotypen vorhersagen zu können, ist die Analyse der Genexpression in frühen Entwicklungsstadien, z.B. über in situHybridisierungstechniken (Kues und Wunder, 1992; Kues et al., 1995a, b). Der Nachteil des Cre/loxP-System und anderer Rekombinasen ist, dass die Methodik relativ aufwendig ist, und in der Regel nur einmal eine Rekombination erfolgt. 2.6.5 Induzierbare Promotoren und konditionale Expressionskontrolle Eine Möglichkeit für die konditionale Expressionkontrolle sind sogenannte induzierbare Promotoren, die mittels exogener Substanzen reguliert werden können. Hierzu gehören der Metallothioneinpromoter (Nottle et al., 1999), sowie Hitzeschock- und Steroid induzierbare Promotoren. Jedoch hat sich gezeigt, dass diese induzierbaren Promotoren in der Transgenese nur beschränkt nutzbar sind, da sie teilweise eine geringe Induktion zeigten, bzw. die Induktoren unspezifische physiologische Effekte zeigten (Yarranton, 1992). Ein wesentlicher Fortschritt wurde mit dem Tetracyclin-regulierten System erzielt (Gossen und Bujard, 1992). Hierbei werden wiederum zwei Transgene in das Genom eingeführt (Abb.5). Ein Konstrukt wird dabei von einem tetracycylin-sensitiven Promoter (ptTA) kontrolliert. Sobald der rekombinante Transaktivator (tTA) an den ptTA bindet, wird das Transgen exprimiert. Der tTA wird von einer zweiten Kassette kodiert; er ist aus zwei Domänen zusammengesetzt: einem Virusprotein 22 (VP22)-Anteil, mit transaktivierender Funktion und einem Protein-Anteil, der Tetracyclin binden kann (Gossen et al., 1995). Durch die Bindung von Tetracyclin kommt es zu einer Konformationsänderung des Gesamtproteins und die DNA-Bindungsfähigkeit geht verloren. In dieser Situation wird der pTA nicht mehr aktiviert und das Zieltransgen abgestellt (tet-offSystem). Durch Mutagenese wurden auch tTA-Varianten entwickelt, die erst durch Tetracyclin-Bindung aktiv an den Promoter binden (Tet-on-system, Furth et al., 1994). In beiden Fällen werden zwei unabhängige Transgene benötigt, was für den Nutztierbereich ein wesentliches Hindernis darstellt. Es gibt aber auch erfolgversprechende Ansätze, beide Transgen-Kassetten auf einem Konstrukt zu verbinden (Schultze et al., 1996) und damit eine vereinfachte konditionale Expression zu erzielen. 18 Abb.5. Konditionale Expressionskontrolle im tet-off-System Für die konditionale Expressionskontrolle werden zwei Konstrukte benötigt. Das tetracycline responsive element (TRE) ist vor einem minimalen Promoter (pminCMV) lokalisiert, dieser wird ohne Aktivierung nicht exprimiert. Der Transaktivator (tTA) wird von dem zweiten Konstrukt kodiert, der tTA bindet an die TRE-Sequenz und aktiviert die Expression. In Gegenwart von Tetracyclin, bzw. Doxycyclin (DOX) wird der tTA durch eine Konformationsänderung inaktiviert, und die Expression des TRE-Konstruktes wird abgestellt. (mod. aus Fa. Clontech Handbuch). 2.7 Genomprojekte und ihre Bedeutung für die Transgenese Seit ca. 1970 werden Methoden zur schnellen Sequenzierung kurzer DNA-Fragmenten entwickelt (Sanger et al., 1973; Maxam und Gilbert 1977; Sanger et al., 1977). Pro Reaktion konnten maximal 400-800 Basen gelesen werden. Aufgrund des notwendigen zeitlichen und technischen Aufwands wurden nur ausgesuchte DNA-Bereiche sequenziert. Vollständig wurden Plasmide und kleine virale Genome mit bis zu 50 Kilobasenpaaren (kBp) sequenziert. Ab ca. 1990 wurde die Komplettsequenzierung des humanen Genoms (HUGO-Projekt) initiiert, zu diesem Zeitpunkt wurden die Kosten für die Sequenzierung einer Base mit ca. 1.US $ angenommen, hochgerechnet für ein haploides Humangenom mit 3 x 10 9 Bp ergibt dies 3 Milliarden US $. Aufgrund der extrem hohen Kosten und der verfügbaren Sequenzierverfahren, die zu dieser Zeit im wesentlichen in „Handarbeit“ durchgeführt 19 wurden, wurde das HUGO-Projekt von vielen Wissenschaftlern zunächst als illusorisch angesehen. Die Entwicklung automatisierter Sequenziermaschinen erlaubte innerhalb weniger Jahre deutlich leistungsfähigere Hochdurchsatzsequenziermethoden und wesentlich reduzierte Kosten (Bennett et al., 2005). Gegenwärtig liegen die Kosten für eine Sequenzierung eines Säugergenoms mit einfacher Abdeckung in der Größenordnung von ca. 100 000 US $. Im Jahr 2001 wurden die ersten Rohentwürfe der humanen Genomsequenzierung publiziert (Lander et al., 2001). Aufgrund der Humangenomsequenz wird die Anzahl der proteinkodierenden Gene heute mit ca. 25 000 annotiert (Goodstadt und Ponting, 2006). Einem Drittel der Gene kann dabei eine eindeutige Funktion zugeordnet, bei einem weiteren Drittel gibt es Vermutungen über die Funktion, und für ein Drittel liegt keine Annotationen vor. Die durch das HUGO-Projekt begonnene Entwicklung führte zur Genomsequenzierung von zahlreichen Organismen. So sind gegenwärtig > 1000 Prokaryoten-Genome und > 49 Eukaryoten-Genome sequenziert. Unter anderem auch die Genome von Rind (Adelson, 2008), Hund (Lindblad-Toh et al., 2005) und Pferd (www.broad.mit.edu.mammals.horse). Für das Schwein liegen Teilgenomsequenzierungen vor. Ein freier Zugang zu umfassenden und annotierten Genominformationen ist über die Internet-Seiten von ENSEMBL (www.ensembl.org), National Center for Biotechnology Information (www.pubmed.org) und der Universität von Californien, Santa Cruz (http://genome.ucsc.edu) möglich. So sind z.B. auf der ENSEMBL-Seite umfassende Genominformationen von 49 Eukaryoten-Spezies, einschließlich von kompletten und Teilgenomsequenzen von Nutztieren aufgearbeitet und direkt zugänglich. Zahlreiche Funktionen können benutzerdefiniert dargestellt und extrahiert werden, z.B. Chromosomenlokalisation annotierter Gene, deren Exon-Intron-Struktur, bekannte Splice-Varianten, Einzelnukleotidpolymorphismen (SNP), die Lokalisation von CpG-reichen-Regionen (CpG-islands), der GC-Gehalt, die Position von repetitiven Elementen. Neben zahlreichen weiteren Informationen kann direkt auf die DNA-Sequenz zugegriffen werden. Noch nicht sequenzierte Bereiche, bzw. Sequenzdaten mit geringer Qualität von Eukaryoten erstrecken sich im wesentlichen auf hochrepetitive Genomabschnitte, z.B. Centromeren, Telomeren, sowie auf das Y-Chromosom. Beispielhaft zeigt Abb. 6 ein 100 kBp-Abschnitt auf dem Rinderchromosom 2, auf dem das Myostatin-Gen (GDF8) lokalisiert ist. Myostatin ist ein negativer Regulator des Muskelwachstums, und eine Reduktion oder der Verlust der Myostatinaktivität führt bei Rindern zum Doppellender-Phänotyp mit Muskelhypertrophie (Grobet et al., 1997; Kambadur et al., 1997; McPherron und Lee, 1997). Bei zahlreichen Fleischrinderrassen, wie Blaue 20 Belgier und Piedmonteser, sind durch konventionelle Züchtung Mutationen und Deletionen im Myostatin-Gen selektioniert worden. Abb. 6. Schematische Darstellung des Genlocus für das bovine Myostatin-Gen (GDF8). Ein 100 kBp-Bereich wurde aus Ensembl (www.ensembl.org) extrahiert. Die Regulation des Muskelwachstums ist bei Säugern offenbar hochkonserviert, so wurde bei Texel-Schafen mit hypertrophen Muskelwachstum eine Mutation im GDF8-Gen entdeckt, die keine Veränderung des Leserasters bedingt, aber im GDF8-Transkript eine falsche Zielsequenz für eine microRNA darstellt. Durch die RNAi-vermittelte Degradation des Transkripts wird eine signifikante Verringerung der Myostatin-Spiegel mit entsprechendem Phänotyp hervorgerufen (Clop et al., 2006). Beim Menschen ist eine Mutation beschrieben worden, die das Spleißen der Prä-mRNA und den Leseraster verändert und ebenfalls zu einem Phänotyp mit Muskelhypertrophie führt (Schuelke et al., 2004). Über ENSEMBL können die entsprechenden syntenen Genomabschnitte dieser Spezies direkt miteinander verglichen 21 werden, bzw. in das Design von genetischen Veränderungen einfließen. Zusammen mit der Verfügbarkeit von cDNA- und genomischen DNA-Klonen zahlreicher Spezies in öffentlichen Genbanken (z.B. Ressourcen Zentrum Berlin, jetzt ImaGenes) ergeben sich für die Transgenese bisher ungeahnte Möglichkeiten, so dass auch komplexe Veränderungen des Genoms (Kuroiwa et al., 2002; Grosse-Hovest et al., 2004) wesentlich vereinfacht werden. 2.8 Epigenetik In der Epigenetik geht es um das Verständnis, wie Information über die Genregulation, die nicht direkt in der DNA-Sequenz kodiert ist, von einer Zell- oder Organismen-Generation in die nächste gelangt (Reik, 2002). Spezifische epigenetische Prozesse umfassen komplexe Genregulations-Mechanismen, unter anderem das Imprinting, das Gen-Silencing und die Reprogrammierung. Die griechische Vorsilbe epi hat mehrere Bedeutungen, wie „nach“, „hinterher“ oder „zusätzlich, über“. So beschreibt die Epigenetik alle Prozesse in einer Zelle, die als „zusätzlich zu“ den Prozessen und Informationen der Genetik gelten. Die Gesamtheit aller epigenetischen Mechanismen wird als Epigenom bezeichnet. Es gibt verschiedene Mechanismen, die in der Epigenetik wirksam werden. Die bestuntersuchten in Säugern sind DNA-Methylierung und Histon-Modifikationen. Bei der DNA-Methylierung wird die Cytosin-Base in CG-Dinukleotiden an der 5-Position methyliert (Abb. 7). Somit wird eine „fünfte“ Base eingeführt, da sich Cytosin und 5-Methyl-Cytosin funktionell unterscheiden. Die Promoterbereiche von vielen Genen umfassen CpG-reiche Regionen, die als „CpG islands“ bezeichnet werden. In aktiven Genen sind die CpG islands unmethyliert, in inaktiven Genen sind die CpG islands und weitere Promoterbereiche häufig Cytosin-methyliert. Auch beim Imprinting ist in der Regel das inaktive Allel an den CpGPositionen Cytosin-methyliert. Während der frühen Embryogenese kommt es zu einem Neusetzen des größten Teils der DNA-Methylierung. So wird in der befruchteten Oozyte zunächst die DNA des paternalen und dann die DNA des maternalen Vorkerns demethyliert. Aufgrund der zeitlichen Aufeinanderabfolge geht man davon aus, dass die Demethylierung des paternalen Vorkerns aktiv und die des maternalen Vorkerns passiv erfolgt (Mayer et al., 2000 a, b). Bis zum Blastozystenstadium erfolgt dann eine aktive Remethylierung. Diese Neusetzung der DNA-Methylierungen wird heute als ein Prozess der Reprogrammierung der Gameten-DNA verstanden, um ein funktionelles Embryonenepigenom zu formen. Ausgenommen von diesen DNA-Methylierungsänderungen im frühen Embryo sind geprägte Gene (imprinted genes), die erst in der Gametogenese neu methyliert werden. 22 Abb. 7. Die DNA-Methyltransferase (DNA-MTase) katalysiert die Methylierung von Cytosin-Basen in CpGDinukleotiden zu 5-Methyl-Cytosin (m5CpG). Dabei dient S-Adenosylmethionin (AdoMet) als MethylgruppenDonor (aus Heby, 1995). Wichtige Histonmodifikationen sind Azetylierungen, Methylierungen oder Phosphorylierungen an spezifischen Aminosäuren der Seitenkette des Histons H3 (Koipally et al., 1999; Senawong et al., 2003; Eilertsen et al., 2008; Nandy et al., 2008). Je nach HistonModifikation kann so ein komprimierter, oder ein aufgelockerter Packungszustand des Chromatins (DNA/Histon-Komplex) erreicht werden (Yao et al., 2001). Im komprimierten Zustand ist die DNA unzugänglich und Transkriptionsfaktoren können nicht oder nur vermindert binden; im aufgelockerten Zustand können Transkriptionsfaktoren leichter binden und die Transkription starten. Aktive DNA-Bereiche mit aufgelockerter DNA werden auch als Euchromatin bezeichnet, komprimierte inaktive DNA-Bereiche als Heterochromatin. Es ist noch nicht eindeutig geklärt, inwieweit CpG-Methylierung und Histon-Modifikationen miteinander interagieren und in welcher Reihenfolge die DNA und Histonmodifikationen erfolgen. Es gilt aber als gesichert, dass beide Mechanismen zum regionalen Packungszustand der DNA (Eu- und Heterochromatin) und zur differentiellen Genaktivität beitragen (Reik, 2002). Dementsprechend sind sie für die Transgenese und die Expressionsprofile in transgenen Tieren von essentieller Bedeutung. 23 3.0 Fragestellungen Die Zielsetzung der vorliegenden Arbeit war es, verbesserte Methoden zur Transgenese von Nutztieren zu etablieren. Dabei wurde insbesondere auf die Spezies Schwein fokussiert, da im Rahmen von BMBF und DFG geförderten Projekten Fördermittel für die Generierung transgener Schweine als Xenotransplantationsmodell eingeworben werden konnten. Methodisch wurden dabei zwei Ansätze verfolgt. Die „klassische“ Mikroinjektion von DNAExpressionskonstrukten in den Vorkern von Zygoten und der somatische Kerntransfer (Klonen) von Kernspenderzellen in entkernte Metaphase II-Oocyten. Für den somatischen Kerntransfer wurden insbesondere die Gewinnung und Kultur der Kernspenderzellen aus adulten und fetalen Geweben untersucht und die Zellkulturen detailliert charakterisiert. Um eine konditionale Expressionskontrolle der eingesetzten DNA-Transgene zu erreichen, wurden neuartige tetracyclin-regulierte Expressionskassetten (tet-off) eingesetzt. Damit sollte die Möglichkeit einer von aussen-regulierbaren Transgenexpression im Schwein evaluiert werden. Bisher war dieser Ansatz in keiner Nutztierspezies untersucht worden. 24 4.0 Ergebnisse und Diskussion Im Ergebnisteil wird auf folgende Aspekte eingegangen: 1) Charakterisierung von primären Kernspenderzellen und Isolierung somatischer Stammzellen (Anger et al., 2003; Kues et al., 2005a, b), 2) Gene knock-down-Versuche in transgenen Embryonen (Iqbal et al., 2007), 3) Generierung und detaillierte Untersuchung von transgenen Schweinen mit konditioneller Genexpression (tet-off: Kues et al., 2006a; Deppenmeier et al., 2006; Niemann u. Kues, 2003; Niemann u. Kues, 2007). Die anderen Aspekte dieser wissenschaftlichen Thematik können in den Nachdrucken der Orginalpublikationen (in Kapitel 10.0) nachgelesen werden (Schätzlein et al., 2004; Hoelker et al., 2005; Yadav et al., 2005; Hornen et al., 2007; Dieckhoff et al., 2007, 2008; Kues u. Niemann, 2004; Niemann et al., 2005; Niemann et al., 2007; Kues et al., 2008). 4.1 Primäre somatische und Stamm-Zellen als Donorzellen für den Kerntransfer Nach der erstmaligen Beschreibung eines erfolgreichen Klonexperiments mit somatischen Zellen (Wilmut et al., 1997), ging man davon aus, dass die Zellzyklusphase der Kernspenderzelle entscheidend für die erfolgreiche Weiterentwicklung der rekonstruierten Embryonen sei. Insbesondere wurde die Ansicht favorisiert, dass primäre Zellen in der G0Phase des Zellzyklus am besten geeignet seien (Wilmut et al., 1997; Wells et al., 1999). Fibroblasten, die direkt aus adultem Gewebe isoliert werden, liegen überwiegend in der G0Phase vor. In der Kultur primärer Zellen wird durch den Zusatz von Serum und Wachstumsfaktoren zum Kulturmedium eine starke Proliferationsstimulation erzeugt (Connell-Crowley et al., 1998), so dass in der Regel maximale Zellwachstums- und Teilungsraten vorliegen. Primäre humane Fibroblasten können in Kultur bis zu 50 Zellteilungen durchführen, bevor sie in Zellseneszenz übergehen (Hayflick, 1965). Für die Anreicherung von Zellkulturen in bestimmten Zellzyklusphasen gibt es eine Reihe von Methoden, die aber meistens für transformierte Zellen optimiert sind (Holley et al., 1968; Reed, 1997; Zetterberg et al., 1995). Für die Zellzyklussynchronisierung primärer Fibroblasten von Nutztierspezies waren keine etablierten Protokolle vorhanden; dies gilt insbesondere für unerwünschte oder toxische Effekte der möglichen Verfahren. Als Kerndonorzellen für den somatischen Kerntransfer wurden hier überwiegend primäre Fibroblastenkulturen angelegt und detailliert charakterisiert. Dabei wurden insbesondere die Homogenität der Fibroblasten, die Proliferationsfähigkeit, die Transfizierbarkeit mit DNA25 Konstrukten und die Zellzyklussynchronisierung untersucht (Kues et al., 2002; Anger et al., 2003; Kues et al., 2005a). Es wurden Methoden für die Isolation primärer Fibroblasten aus fetalen und adulten Gewebe von Nutztieren entwickelt und die etablierten Zellkulturen hinsichtlich der Proliferationsfähigkeit in Kultur und der Zellzyklussynchronisierung detailliert untersucht. Die primären Zellen konnten genetisch verändert werden (Dieckhoff et al., 2007) und wurden erfolgreich für Klonversuche verwendet (Schaetzlein et al., 2004; Hornen et al., 2007; Dieckhoff et al., 2008). Es konnte gezeigt werden, dass porcine fetale Fibroblasten grundsätzlich Zellzyklus synchronisierbar sind. Maximal wurden Anreicherungen in der G0/G1-Phase des Zellzyklus von 80-90 % der Zellen erreicht, dabei wurden sowohl Serumreduktion als auch chemische Inhibitoren des Zellzyklus (Pedrail et al., 1980; Kitagawa et al., 1994), wie Aphidicolin und Butyrolactone eingesetzt (Kues et al., 2000). Die optimale Zellzyklussynchronisierung wurde nach 48 Stunden Kultur in serumreduzierten Zellkulturmedien erreicht; damit sind wesentlich kürzere Synchronisationszeiten möglich, als zunächst beschrieben (Wilmut et al., 1997; Wells et al., 1999). Bei längeren Serumentzugszeiten (als 48 Stunden) kam es zunehmend zum apoptotischen Zelltod, wobei eine direkte Korrelation mit der Serumreduktion und der Zeitdauer der Behandlung festgestellt werden konnte. In den primären Fibroblasten wurde dabei eine unkonventionelle Form der Apoptose beobachtet (Kues et al., 2002). Die Messung der Zellzyklusphasen durch Fluorescence activated cell sorting (FACS) wurde durch Reverse transcriptase-polymerase chain reaction (RT-PCR)-Bestimmungen der Transkripte des zellzyklus-spezifisch regulierten Gens Polo-ähnlicher Kinase (Polo-like kinase) auf molekularer Ebene untermauert (Kues et al., 2000; Kues et al., 2002). In den Primärkulturen porciner Fibroblasten wurde ein neuer Stammzelltyp gefunden, der mit Hilfe der Explantatmethode und der Kultur unter hohen Zelldichten angereichert werden konnte (Abb. 8). Diese fetalen somatischen Stammzellen (FSSCs) konnten aus fetalem Bindegewebe von Schwein, Maus, Rind und Schaf isoliert werden (Kues et al., 2005a), in adultem Gewebe kommen sie offenbar in wesentlich geringerer Frequenz vor. 26 Tissue explant Fetus reseeding of outgrowing cells Standard culture Fibroblast monolayer confluent, mitotic inactive High density culture Colonies of FSSCs colony growth on monolayer, mitotic active, Oct-4, Stat3, TNAP positive Suspension culture Anchorage independent growth reaggregation, growth as spheroids Abb. 8. Isolation fetaler somatischer Zellen (FSSC, aus Kues et al., 2005b). Die FSSCs wachsen als Kolonien auf einem autologem Nährzellrasen aus Fibroblasten, sie exprimieren Stammzellmarker (Yeom et al., 1996), wie OCT4, alkaline Phosphatase (AP) und STAT3, zeigen kein Hayflick-Limit und nach Injektion muriner FSSCs in Maus-Blastozysten konnten chimäre Feten erhalten werden, in denen die injizierten Zellen zu somatischen Geweben und der Keimbahn beitrugen (Abb.9). Inwieweit der beobachtete Chimärismus durch Zellfusion (Ying et al., 2002; Wang et al., 2003) bedingt ist, muss in weitergehenden Untersuchungen abgeklärt werden. Porcine FSSCs wurden zudem erfolgreich im Kerntransfer eingesetzt (Hornen et al., 2007). Aufgrund ihrer höheren Potenz sind FSSCs möglicherweise bessere Kernspender im Klonverfahren. Durch ihre längere Proliferationsfähigkeit in Kultur vereinfachen sie genetische Modifikationen und die Isolierung von Zellklonen. Zur Zeit wird davon ausgegangen, dass FSSCs einen gewebe-spezifischen Stammzelltyp darstellen. Fibroblasten stellen einen Hauptzelltyp in Bindegewebe dar, wo sie eine Reihe von Funktionen erfüllen (Moulin et al., 1999; Chang et al., 2002; Han et al., 2002), unter anderem sind sie an der Regeneration beteiligt. In Fetalstadien von Säugern verheilen Hautverletzungen ohne Narbenbildung (Moulin et al., 1999), was als ein indirekter Hinweis auf eine höhere Zellplastizität von Bindegewebszellen in diesem Stadium gewertet werden kann. 27 A B Wt chimeric fetus day 15.5 p.c. Chimeric fetuses control OG2/Rosa26 Abb.9. Zellchimärismus in Mausfeten nach Injektion von Rosa26/OG2-transgenen FSSCs in Blastozysten. A) Durch histologischen Nachweis der LacZ-Expression (Pfeile) kann die Beteiligung der injizierten Zellen an den Fetalorganen gezeigt werden (rechter Embryo). Links ein Kontrollembryo. B) Nachweis von GFP-positiven Zellen (OG2) in chimärer Keimleiste. Rechts als Positivkontrolle die Keimleiste eines OG2 transgenen Fetuses (aus Kues et al., 2005a). 28 4.2 Gene knock-down Versuche in transgenen Embryonen Das Transkriptom in frühen Säugerembryonen wird zunächst durch maternale mRNAs geprägt, die in der Oozyte angereichert vorliegen (Schultz, 2002). Im Zygotenstadium erfolgt keine Transkription des embryonalen Genoms, die Proteinsynthese ist in diese Phase von den maternalen Transkripten abhängig. Die Aktivierung des embryonalen Genoms (embryonic genome activation) erfolgt spezies-spezifisch bei murinen Embryonen am Ende des Zygotenstadiums, bei humanen und porcinen Embryonen im 4-Zellstadium und bei bovinen Embryonen im 8-Zellstadium (Telford et al., 1990). In der Literatur war gezeigt worden, dass der RNAi-Mechanismus in Säugerembryonen funktionell ist, dabei wurden überwiegend lange doppelsträngige RNA (500-1000 bp) injiziert, und verschiedene Gentranskripte, wie E-Cadherin, Mos, Plat, Oct4, Coxa, Cox5b und Cox6b selektiv degradiert (Svoboda et al., 2000; Wianny et al., 2000; Plusa et al., 2004; Paradis et al., 2005; Nganvongpanit et al., 2007; Cui et al., 2006). Die doppelsträngigen RNAs werden durch das zelleigene Enzym Dicer in siRNAs prozessiert, die dann in dem eigentlichen RNAi-Mechanismus den Abbau von komplementären Transkripten verursachen (Svoboda et al., 2000). Bei diesem Verfahren ist also eine relativ hohe Beladung der Embryonen mit siRNAs notwendig, von denen nur ein Teil funktionell ist, zusätzlich kann es zu unspezifischen Effekten kommen. Zudem kann bei Genen, die ein biphasisches Expressionsmuster zeigen, d.h. die sowohl im maternalen Transkriptom vorliegen, als auch im embryonalen Genom transkribiert werden, nicht ausgeschlossen werden, dass maternale und embryonale Transkripte unterschiedlich sensitiv für den RNAi-Mechanismus sind. So ist bekannt, dass maternale Transkripte eine Halbwertzeit im Bereich von Tagen, embryonale Transkripte dagegem im Bereich von Minuten besitzen (Karlson et al., 2005). In transgenen Mäusen wurde getestet, ob im RNAi-Mechanismus eine differentielle Degradierung von maternalen und embryonalen Transkripten auftritt. Dazu wurde eine funktionelle synthetische siRNA (22 bp) gegen GFP in transgene Mausembryonen injiziert. (Iqbal et al., 2007). Um die Degradation von maternalen und embryonalen Transkripten verfolgen zu können, wurden transgene Mäuse (OG2), die das Green Fluorescent Protein (GFP) unter Kontrolle des keimbahnspezifischen Promoters Oct4 exprimieren (Szabo et al., 2002), verwendet. In transgen-homozygoten Embryonen wird GFP in allen Blastomeren exprimiert, also sowohl maternal als auch embryonal. Bei Verpaarungen mit Wildtypmäusen entstehen hemizygot-transgene Embryonen, bei denen die GFP-Expression davon abhängt, ob sie das Transgen-Allel von der mütterlichen (OG2mat/-) oder der väterlichen (OG2pat/-) Seite 29 geerbt haben. OGmat/- Embryonen zeigen eine kontinuierliche GFP-Expression von der Oocyte bis zur Blastozyste, während OGpat/--Embryonen erst ab dem 4-8-Zellstadium GFP-positiv werden (Abb.10). Die RNAi der GFP-Expression kann somit an lebenden Embryonen selektiv für maternale und embryonale Transkripte evaluiert werden. Abb. 10. Vererbungsabhängige Expression von Oct4-GFP in hemizygoten Mausembryonen. A) Embryonen mit paternal vererbten Transgen (OG2pat/-) zeigen GFP-Expression ab dem 8-Zellstadium, Embryonen mit maternal vererbten Transgen (OG2mat/-) zeigen in allen Stadium von Zygote bis Blastocyste GFP-Fluoreszenz. B) Schematischer Aufbau der siRNA, die verwendete siRNA ist mit dem Fluorochrom Rhodamine konjugiert. C) Injektion der siRNA in OG2mat/--Zygote unter Hellfeldbedingungen (oben), Fluoreszenzanregung für GFP (mitte) und Fluoreszenzanregung für Rhodamin (unten) (aus Iqbal et al., 2007). Durch Mikroinjektion einer short interfering RNA (siRNA), die gegen die GFP mRNA gerichtet ist, wurden die unterschiedlichen RNAi-Kinetiken bei Embryonen mit maternaler, bzw. embryonaler GFP-Expression ermittelt. Dabei zeigte sich, dass die Expression vom paternalen Allel nahezu komplett inhibiert werden kann; es war keine GFP-Fluoreszenz nachweisbar. In den OGmat/- Embryonen konnte eine Reduktion der GFP-mRNA um ca. 95% nachgewiesen werden, aufgrund der hohen Proteinstabilität von GFP war aber erst in Blastozysten eine relative Verringerung der GFPFluoreszenz feststellbar (Abb.11). Diese Untersuchung zeigt, dass eine einmalige Injektion von siRNA im Zygotenstadium ausreichend ist, um bis zum Blastozystenstadium eine RNAInterferenz zu erreichen. Durch die Verwendung einer synthetischen siRNA, können unspezifische Effekte, die bei langen doppelsträngigen RNAs beobachtet werden, weitgehend 30 vermieden werden. Um zu bestimmen, wie lange der Effekt einer einmaligen siRNAInjektion, anhält, müssten in Folgeuntersuchungen Postimplantations-Stadien untersucht werden. (OG2pat/-) (OG2mat/-) Abb.11. RNAi in hemizygoten Mausembryonen. Nach Injektion der GFP-spezifischen siRNA (Fig.10) in hemizygote OG2 transgene Zygoten wurden diese in vitro kultiviert. 2-Zell, Morula- und Blastozystenstadien von OG2pat/- (links) und OG2mat/- (rechts) sind im Hellfeld und unter GFP-Fluoreszenzanregung dargestellt (aus Iqbal et al., 2007). Im Gegensatz zu den relativ aufwendigen Verfahren eine RNA-Interferenz in postimplantatorischen Embryonen zu induzieren (Mellitzer et al., 2002; Caligari et al., 2004; Soares et al., 2005), ist das siRNA-Injektionsverfahren wesentlich einfach durchzuführen und zeitlich mindestens bis zum Blastozystenstadium effektiv. 31 4.3. Generierung transgener Schwein mit konditioneller Transgenexpression Über die DNA-Mikroinjektion in einen Vorkern von Schweinezygoten wurden transgene Linien mit Expression des humanen Komplementregulators CD59 erstellt (Niemann und Kues, 2003). Als regulative Sequenz wurde der Cytomegalovirus (CMV) Immediate EarlyPromotor gewählt, der als einer der stärksten Promotoren in der Zellkultur gilt. Abb. 12. Generation transgener Schwein mit Expression von hCD59 A) Minigen-Konstrukt mit hCD59 cDNA, B) Injektion des aufgereinigten Konstruktes in einen Vorkern einer Schweinezygote, C) Transgenes F0-Tier, D) Organspezifische Expression von hCD59 in einem F1-Tier, E) Nachweis von hCD59 auf der Zelloberfläche von porcinen Fibroblasten und Endothelzellen, F) funktioneller Nachweis von hCD59 in Schweinefibroblasten durch verminderte Lyse durch Humankomplement, G) funktioneller Nachweis von hCD59 in einem Nierenperfusionsystem mit Humanblut (aus Niemann und Kues, 2003). Es wurden 5 Linien transgener Schweine mit diesem Konstrukt etabliert. Abb. 12 fasst die Ergebnisse aus der Linie 5 zusammen. Bei Expressionsuntersuchungen zeigte sich, dass eine 32 starke Expression von hCD59 in Pankreas, Niere, Herz und Haut vorlag. Auch primäre Endothelzellen, die aus der Aorta isoliert wurden, wiesen eine, wenn auch geringe Expression von hCD59 auf. In Perfusionen von Schweinenieren mit Humanblut, die in Zusammenarbeit mit der Medizinischen Hochschule Hannover durchgeführt wurden, konnte gezeigt werden, dass die vorhandene hCD59-Expression ausreichte, um wesentlich verbesserte Perfusionszeiten zu erreichen und die hyperaktute Abstoßung zu unterdrücken (Niemann et al., 2001). Feingewebliche Untersuchungen zeigten, dass eine starke Variegation der hCD59-Expression in allen CMV-transgenen Linien vorlag (Abb. 13). So waren in der Niere einzelne Zellen stark anfärbbar, während benachbarte Zellen keine nachweisbare hCD59-Expression aufwiesen. H Abb. 13. Variegierte Transgenexpression in CMV-hCD59 transgenen Schweinen (L5). A), C), E) immunhistologischer Nachweis von hCD59 in Herz, Pankreas und Nierengewebe eines Linie 5Tieres. B), D) und F) Kontrollgewebe eines wt-Tieres (aus Niemann et al., 2001). H) Vergrößerter Ausschnitt einer immunhistologischen Anfärbung für hCD59 in der Niere. In einem weitergehenden Ansatz wurde versucht, diese Limitationen zu überwinden, dazu wurde eine neuartige Tetracyclin-regulierbare Expressionskassette (NTA) zusammen mit der Arbeitsgruppe von Dr. Hauser (GBF/jetzt Helmholtz-Zentrum für Infektionsforschung) eingesetzt. Tetracyclin-regulierbare Expressionssysteme (tet-off, bzw. tet-off) wurden für die konditionale Genregulation in Zell-Linien und transgenen Mäusen entwickelt (Gossen und Bujard, 1992; Furth et al., 1994; Lewandowski, 2001; Corbel und Rossi, 2002), um die Limitationen zu überwinden, die mit der konditionalen Genregulation auf der Basis von Metall- oder Steroid-sensitiven Promotoren bestanden (Mayo et al., 19982; Lee et al., 1981; Miller et al., 1989; Pursel et al., 1989). Das originale Tet-System besteht aus zwei Expressionskassetten, einem Transaktivator-Konstrukt und einem Transaktivator-regulierten 33 Konstrukt, die in zwei Mauslinien etabliert und dann durch Verpaarung in einem Genom kombiniert werden (Lewandowski, 2001). Da der Zeitaufwand für die Verkreuzung von zwei Nutztierenlinen wesentlich höher ist als bei der Maus, wurde hier ein Ansatz gewählt, der beide funktionellen Elemente auf einem Konstrukt vereinigt und somit nur die Erstellung einer transgene Linie erfordert (Kues et al., 2006a). Dazu wurde ein bicistronisches Konstrukt entworfen (Abb. 14), bei dem eine mRNA gebildet wird, die einen Komplementregulator (hCD59 oder hDAF) und den Tet-Transaktivator (NTA-RCA) kodiert. Durch die basale Expression des Konstruktes kommt es dann zu einer autoregulativen Hochregulation der Expression; die Transgenexpression ist durch Zugabe von Tetracyclin oder Doxycyclin abstellbar, bzw. regulierbar (Abb. 14). Als eigentliche Transgene wurden die humanen Komplentregulatoren CD59, bzw. DAF jeweils in das erste Cistron kloniert. Es wurden also zwei NTA-Konstrukte mit den Kombinationen CD59-tTA und DAF-tTA verwendet. Der Vorteil dieser Konstrukte gegenüber den Orginalsystem (Gossen und Bujahr, 1992) besteht darin, dass nur ein Transgenkonstrukt in das Genom eingebracht wird und die Verpaarung von zwei Linien entfällt. Dox Inactivated exogenous Dox RCA Transactivator (AAA)n + RCA IRES tTA pA ptTA Tet-O TATA box Abb. 14. Autoregulative bicistronische Expressionskassette (NTA). Der tetracyclin-regulierte Promoter besteht aus sieben Tet-Operator-Sequenzen und einem minimalen Promoter mit einer TATA-Box. Die bicistronische Kassette wird durch eine IRES-Sequenz getrennt, das erste Cistron kodiert einen RCA (entweder CD59 oder DAF) und das zweite Cistron den Transaktivator. Nach Transkription führt der tTA durch Bindung an den eigenen Promoter zu einer autoregulativen Hochregulation, durch Zugabe von Doxycyclin wird der tTA inaktiviert (verändert aus Kues et al., 2006a). Die Funktionalität der NTA-Kassette wurde durch Transfektion in murine NIH-3T3 Zellen und in porcine Swine testis epithelium (STE)-Zellen getestet Dabei konnte sowohl in 34 Zellpopulationen als auch in Einzelzellklonen eine volle Aktivität der NTA-Kassette beobachtet werden, dass heisst durch basale Transkription konnte eine Hochregulation der humanen Komplementregulatoren CD59, bzw. DAF erzielt werden. Die Transgenexpression war durch Tetracyclin-Supplementation des Zellkulturmediums reversibel abstellbar (Kues et al., 2006a). Insgesamt wurden 10 transgene Linien (hemizygot) von Schweinen mit diesem Konstrukt über Injektion der linearisierten DNA in Zygotenvorkerne erstellt und charakterisiert (Tab. 3; Kues et al., 2006a, Niemann und Kues, 2007). Dabei zeigte sich, das in den hemizygoten Linien die NTA-Kassette nahezu komplett stillgelegt vorlag (gene silencing); also trotz Vorhandenseins des Transgens im Genom keine Transkription erfolgte. Gene silencing wird auch in Mauslinien beobachtet, dass allerdings 10 unabhängige Linien mit unterschiedlichen Kopienzahlen des Transgens (1-3) und unterschiedlichen Integrationsorten eine (nahezu) komplettes gene silencing aufwiesen, deutet daraufhin, dass entweder das Konstrukt selbst diesen Effekt hervorrief, oder in Schweinen deutlich sensitivere Mechanismen zum Erkennen und Stilllegen von Fremd-DNA als in Mäusen aktiv sind. Die Expression der NTA-Kassette konnte in hemizygoten Tieren nur in der Skelettmuskulatur gefunden werden, und zwar waren offenbar zufallsmäßig einzelne Fasern in der feingeweblichen Untersuchung Transgen-positiv (Abb. 15B). Auffallend war, dass dieses Muster bei 9 von den untersuchten Linien vorlag (Tab.3), linienabhängig waren 5-20% der Muskelfasern hCD59 oder hDAF positiv. Die positiven Fasern waren dabei durchgehend stark positiv angefärbt (Kues et al., 2006a). Tab. 3: Transgenexpression in Schweinen mit einer und zwei NTA-Kassetten Transgene Schweine mit einer NTA-Kassette DAF-NTA Herz Leber Lunge Niere Pankreas Skel. Muskel Thymus Lymphozyten Fibroblasten Transgene Schweine mit zwei NTA-Kassetten CD59-NTA DAF-NTA x CD59-NTA L12 L13 L14 L15 L16 L17 L19 L20 L21 L22 L13 x L20 (n = 7) DAF CD59 L15 x L20 (n = 2) DAF CD59 - ++ - + - + - + - + - + - + - + - ++ + n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. ++ - n.d. n.d +* - - n.d. - n.d. n.d. n.d. n.d. n.d. - n.d. n.d . - ++* + n.d. n.d. + ++ + + ++ ++++ ++ Zusammengefasste Expressionsdaten aus Northern blot, Immunhistologie und FACS-Messungen; (+)-Zeichen zeigen Expressionshöhe an, (-) keine Expression detektierbar, (n.d.) nicht untersucht und (*) nach Mitogenstimulation. Primäre Fibroblasten aus hemizygoten Tieren exprimierten kein Transgen, auch nach Transfektion mit einem tTA-Konstrukt wurde keine Expression gefunden. Entsprechende 35 Befunde wurden nach Injektion des tTA-Plasmids in die Skelettmuskulatur gemacht. Dagegen konnte in Mitogen-stimulierten Lymphozyten die Expression von hCD59 und hDAF eindeutig gezeigt werden (Kues et al., 2006a). Durch Fütterung mit Doxycyclin war die Expression in den Muskelfasern abgestellbar. Durch Resequenzierung der Konstrukte aus genomischer DNA der transgenen Tiere, konnte ausgeschlossen werden, dass die beobachteteten Expressionsmuster durch Deletion von regulativen Sequenzen hervorgerufen wurden. Ansätze, die transgenen Linien auf Transgen-Homozygotie zu verpaaren, waren nicht erfolgreich. Offenbar aufgrund eines bereits hohen Inzuchtgrades im vorhandenen Bestand führten Geschwisterverpaarungen zu sehr kleinen Würfen (0-3 Ferkel) mit lebensschwachen Ferkeln. Daher wurden Tiere aus Linien mit NTA-CD59 und NTA-DAF-Transgenen verpaart. In den normal großen Würfen wurden in etwa die erwarteten Verhältnisse von 25 % Nachkommen mit beiden Kassetten, 50% mit einer Kassette und 25% Wildtyp gefunden (Kues et al., 2006a). Bei Tieren mit zwei NTA-Kassetten wurde linien-abhängig die Hochregulation von einer oder beider Kassetten gefunden (Tab. 3; Kues et al., 2006a; Niemann und Kues, 2007). Dabei kam es einer Ausweitung der exprimierenden Zellen, so wurden regelmäßig bis zu 100 % positive Skelettmuskelfasern gefunden, und in zahlreichen anderen Organen, wie Leber, Haut, Herz und Niere lagen positive Zellen vor (Abb. 15A, C). In den NTA-doppelttransgenen Tieren wurde eine Doxycyclin-abhängige Transgenexpression gefunden (Abb. 16). Nach oraler Applikation von 3.3 mg Doxycyclin pro kg Lebendgewicht und Tag wurde bereits nach 2 Tagen eine massive Reduktion der bicistronischen Transkripte gefunden. In Abb. 16 wurden von drei doppelt-transgenen Geschwistertieren eines mit einem Placebo und zwei mit Doxycyclin gefüttert. Die gewählte Doxycyclin-Dosis von 3.3 mg/ Kilogramm Lebendgewicht und Tag wurde aufgrund der Literaturangaben für die Doxycyclin-Transgenregulation bei Mäusen (Furth et al., 1994; Kistner et al., 1996; Zhu et al., 2001) extrapoliert; sie liegt deutlich unter der für Schweine empfohlenen antimikrobiell wirksamen Dosis von 12 mg/kg und Tag. Die Transgenexpression in transgenen Schweinen blieb nach dem Absetzen der Doxycyclin-Gabe für mindestens 50 Tage abgeschaltet, bevor eine Reexpression messbar ist (Abb. 16). Eine mögliche Erklärung ist, dass Doxycyclin offenbar effektiv in Knochen zwischengespeichert und dann zeitverzögert in den Kreislauf zurückgeführt werden kann (Stepensky et al., 2003). In transgenen Mäusen wurden unterschiedliche Wirksamkeiten von Doxycyclin in Abhängigkeit vom Mausstamm beschrieben (Robertson et al., 2002). In nachfolgenden Dosis-Wirksamkeitsuntersuchungen 36 bei doppelttransgenen Schweine wurde die zweimalige Gabe einer Doxycyclin-Dosis von 0,6 mg/kg als ausreichend für eine sichere Expressionsregulation gefunden (nicht gezeigt). Abb. 15. Transgen-Expression in einfach und doppelttransgenen Schweinen (NTA). A) Northern blot mit CD59-Sonde; (1, 2, 3), Muskelbiopsien von NTA-CD59/NTA-DAF doppelttransgenen Tieren; (4,5,6), Muskelbiopsien von NTA-CD59 einzeltransgenen Tieren; (7, 8) wt-Kontrollen; (9, 10) PositivKontrollen von CMV-CD59 transgenen Tieren. In den Proben aus einzeltransgenen NTA-Tieren (4, 5, 6) ist die CD59-Expression erst nach längerer Expositionszeit sichtbar. B) Immunhistologischer Nachweis von CD59 und DAF in einzeltransgenen Tieren (NTA) im Skelettmuskel. Einzelne Muskelfasern sind angefärbt. Oberer Reihe Muskellängsschitte, untere Reihe Muskelquerschnitte. In anderen Organen wurden keine Zellen mit Transgenexpression gefunden. C) Immunhistologischer Nachweis von CD59 und DAF in doppeltransgenen Tieren. In doppeltransgenen Tieren aus Linie 13 mit Linie 20-Verpaarungen ist die Muskulatur komplett positiv für CD59 gefärbt (M.) in Leber (Le.), Lunge (Lu.) und Pankreas (P.) sind einzelne Zellen positiv. Dagegen ist DAF nur in einzelnen Muskelfasern nachweisbar. In doppelttransgenen Tieren aus der Verpaarung L15 mit L20 (unterster Reihe, M.) ist die Expression von CD59 und DAF auf einzelne Muskelfasern beschränkt, wobei einige Faser nur CD59Expression aufweisen (Pfeile, bzw. gestrichelte Kreise) (mod. aus Kues et al., 2006a). Die Analyse auf DNA-Methylierung durch Bisulfitsequenzierung in Promoterbereichen der NTA-Konstrukte ergab, das einzeltransgene Tiere eine nahezu komplette Methylierung an CpG-Dinukleotiden aufwiesen, während in den Promoterbereichen der doppeltransgenen Tieren eine ca. 50% Reduktion der Methylierung gefunden wurde. In Zellklonen, Maus 3T3 und porcinen STE-Zelllinien, waren die Promotorsequenzen methylierungsfrei. Offenbar wird die NTA-Kassette während der Ontogenese stillgelegt (gene silencing); dieses manifestiert 37 sich einer DNA-Methylierung der Promotorsequenzen. In den doppelttransgenen Tieren kann dieses Silencing unterdrückt werden; offenbar kann durch den autoregulativen Charakter der Expressionskontrolle das Silencing verhindert werden. Möglicherweise geschieht dies direkt durch die Bindung des tTA, was in Abhängigkeit von der basalen Expressionshöhe ein Silencing bestimmter Integranten dauerhaft verhindern würde. Gegenwärtig laufen Versuche, durch weitere Verpaarung mehr als zwei NTA-Kassetten in einem Tier zu kombinieren. Das hier gefundene bimodale Expressionsmuster wurde mittlerweile auch in Zell-Linien für bicistronische Konstrukte beschrieben (May et al., 2008). Abb. 16. Konditionale Transgenexpression im Schwein Drei NTA-doppelttransgenen Geschwistertieren wurde über 6 Tage ein Placebo oder Doxycyclin (3.3 mg/kg) gefüttert. Zu verschiedenen Zeitpunkten wurden Muskelbiopsien genommen und im Northern blot auf die Expression von hCD59, hDAF und des porcinen ß-Actin (ACTB)-Gens untersucht (aus Kues et al., 2006a). Die Bedeutung der NTA-transgenen Tiere liegt im wesentlichen in den neuen Einblicken in die Regulation von Gensequenzen im Säugergenom. In diesem Tiermodell konnte ein stillgelegtes Transgen erstmals wieder reaktiviert werden. Durch die Verpaarung zu doppelttransgenen Tieren wird offenbar in einem kritischen Entwicklungsfenster das Silencing der Transgene verhindert. Die detaillierte Identifikation der beteiligten Mechanismen und zeitlichen Abläufe in weitergehenden Untersuchungen wird für eine optimierte Transgenese, insbesondere in Nutztieren von entscheidender Bedeutung sein. 38 5.0 Zusammenfassung In der vorliegenden Arbeit wurden experimentelle Ansätze zur Transgenese von Nutztieren durchgeführt. Aus primären Fibroblastenkulturen von Nutztieren wurden Stammzellen (FSSCs) abgeleitet, die wesentliche Vorteile für eine genetische Modifikation haben und die Effizienz des somatischen Kerntransfers erhöhen können. In transgenen Embryonen wurde mit einer synthetischen siRNA ein allel-spezifischer Gene knock-down gezeigt. Es wurden transgene Schweine mit konditionaler Expressionskontrolle (tet-off) erstellt und detailliert charakterisiert. Dabei zeigten Tiere aus den hemizygoten Linien ein nahezu komplettes Silencing der Transgen-Kassetten. Durch die Verpaarung zu doppelttransgenen Tieren konnten die Transgenkassetten reaktiviert werden. Dabei konnten die NTA-Kassetten von allen vier untersuchten Integrationsorten reaktiviert werden, die Reaktivierung war dabei in linien-spezifischen Kombinationen möglich. Damit wurde erstmalig eine wirksame konditionale Expressionskontrolle im Nutztier gezeigt; und es wurde nachgewiesen, dass epigenetische Mechanismen, insbesondere die DNA-Methylierung, mit der konditionalen Expression interferieren können. 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Lemme und Frau K. Korsawe und die Grosstierversuche durch Herrn K.-H. Hadeler technisch hervorragend unterstützt. Für die photo- und printtechnische Unterstützung wird dem Photographen gedankt. Für das zur Verfügungstellen von spezifischen Plasmiden und Zell-Linien wird Dr. M. Anger, (Universität Oxford), Dr. J. Bode (HZI, Braunschweig), Dr. M. Buchholz (MPI, Dresden), Dr. T. Cantz (MPI, Münster), Dr. K. Collins (Berkeley, Californien), Dr. E. Heinemann (Whitehead Institute, Cambridge), Dr. Lipps, (Universität Witten), Dr. U. Martin (MHH, Hannover), Dr. Ritter (Charite, Berlin), Dr. T. Saric (Universität Köln), Dr. V. Witzemann (MPI, Heidelberg) und Dr. J.K. Zhu (University of California) ganz herzlich gedankt. Für die finanzielle Förderung der DFG (Transregio 535) und des BMBF wird gedankt. 54 8.0 Darstellung des eigenen Anteils an den wissenschaftlichen Publikationen Characterisierung primärer Zellen als Donorzellen für den Kerntransfer Anger, M., Kues, W.A., Klima, J., Mielenz, M., Motlik, J., Carnwath, J.W. and Niemann, H. 2003 Cloning and cell cycle-specific regulation of Polo-like kinase in porcine fetal fibroblasts. Molecular Reproduction and Development 65, 245-253. a) Idee und Versuchsplanung b) Versuchsdurchführung c) Auswertung d) Verfassen des Manuskriptes Anger, M, Kues, W.A. Anger, M., Kues, W.A., Klima, J. Mielenz, M. Anger, M., Kues, W.A. Anger, M., Kues, W.A., Motlik, J., Niemann, H. Kues, W. A., Petersen, B., Mysegades, W., Carnwath, J.W. and Niemann, H. 2005a. Isolation of fetal stem cells from murine and porcine somatic tissue. Biology of Reproduction 72, 1020-1028 a) Idee und Versuchsplanung b) Versuchsdurchführung c) Auswertung d) Verfassen des Manuskriptes Kues, W.A. Kues, W.A., Mysegardes, W. Kues, W.A., Niemann, H. Kues, W.A., Carnwath, J.W., Niemann, H. Yadav, P., Kues, W.A., Herrmann, D., Carnwath, J.W., Niemann, H. 2005. Bovine ICMs express the Oct4 ortholog. Molecular Reproduction and Development 72, 182-190. a) Idee und Versuchsplanung b) Versuchsdurchführung c) Auswertung d) Verfassen des Manuskriptes Kues, W.A., Niemann, H. Yadav, P. Kues, W.A. Yadav, P., Kues, W.A., Niemann, H. Dieckhoff B, Karlas A, Hofmann A, Kues WA, Petersen B, Pfeifer A, Niemann H, Kurth R, Denner J. 2007. Inhibition of porcine endogenous retroviruses (PERVs) in primary porcine cells by RNA interference using lentiviral vectors. Archives of Virology 152, 629-34. a) Idee und Versuchsplanung b) Versuchsdurchführung c) Auswertung d) Verfassen des Mauskriptes Denner, J Dieckhoff, B. Dieckhoff, B., Kues, Denner, J. Denner, J; Kues, W.A.; Denner, J., Niemann, H. 55 DNA-Mikroinjektion und somatischer Kerntransfer Schätzlein S., Lucas-Hahn, A., Lemme, E., Kues, W.A., Dorsch, M., Manns, M., Niemann, H., Rudolph, K.H. 2004. Telomere length is reset during early embryogenesis. Proceedings of the National Academy of Sciences of the USA 101, 8034-8038. a) Idee und Versuchsplanung b) Versuchsdurchführung c) Auswertung d) Verfassen des Manuskriptes Niemann, H., Rudolph, K.H. Schätzlein, S., Lucas-Hahn, A., Lemme, E., Kues, W.A. Rudolph, K.H., Schätzlein, S., Kues, W.A., Niemann, H. Rudolph, K.H., Schätzlein, S., Kues, W.A., Niemann, H. Hölker, M., Petersen, B., Hassel, P., Kues, W.A., Lemme, E., Lucas-Hahn, A., Niemann, H. 2005. Duration of in vitro maturation of recipient oocytes affects blastocyst development of cloned porcine embryos. Cloning and Stem Cells 7, 34-43. a) Idee und Versuchsplanung b) Versuchsdurchführung c) Auswertung d) Verfassen des Manuskriptes Niemann, H., Kues, W.A.. Hölker, M., Petersen, B. Hölker, M.., Kues, W.A., Niemann, H. Hölker, M.; Kues, W.A., Niemann, H. Kues, W.A., Schwinzer, R., Wirth, D., Verhoeyen, E., Lemme, E., Herrmann, D., Barg-Kues, B., Hauser, H., Wonigeit, K., Niemann H. 2006 Epigenetic silencing and tissue independent expression of a novel tetracycline inducible system in double-transgenic pigs. FASEB J. 20, E1-E10, doi: 10.1096/fj.05-5415fje. a) Idee und Versuchsplanung b) Versuchsdurchführung c) Auswertung d) Verfassen des Manuskriptes Kues, W.A., Niemann, H., Hauser, H., Wonigeit, K. Kues, W.A., Schwinzer, R. Kues, W.A., Schwinzer, R. Kues, W.A., Niemann, H. Deppenmeier, S., Bock, O., Mengel, M., Niemann, H., Kues, W., Lemme, E., Wirth, D, Wonigeit, K., Kreipe, H. 2006. Health status of transgenic pig lines expressing human complement regulator protein CD59. Xenotransplantation 13, 345-356. a) Idee und Versuchsplanung b) Versuchsdurchführung c) Auswertung d) Verfassen des Manuskriptes Wonigeit, K., Niemann, H., Kreipe, H. Deppenmeier, S. Deppenmeier, S., Kues, W.A., Bock, O., Wonigeit. K Deppenmeier, S., Bock, O., Kues, W.A., Niemann, H. Hornen, N., Kues, W.A., Carnwath, J.W., Lucas-Huhn, A., Petersen, B., Hassel, B., Niemann, H. 2007. Production of viable pigs from fetal somatic stem cells. Cloning and Stem Cells 9, 364-73. a) Idee und Versuchsplanung b) Versuchsdurchführung c) Auswertung d) Verfassen des Manuskriptes Kues, W.A., Niemann, H. Hornen, N. Hornen, N., Kues, W.A. Hornen, N., Niemann, H., Kues, W.A. 56 Iqbal, K, Kues, W.A., Niemann, H. 2007. Parent of origin dependent gene-specific knock down in mouse embryos. Biochemical Biophysical Research Communications 358, 727-732. a) Idee und Versuchsplanung b) Versuchsdurchführung c) Auswertung d) Verfassen des Manuskriptes Kues, W.A. Iqbal, K. Iqbal, K, Kues, W.A. Kues, W.A., Niemann, H. Dieckhoff, B., Petersen, B., Kues, W.A., Kurth, R., Niemann, H., Denner, J. 2008. Knockdown of porcine endogenous retrovirus (PERV) expression by PERV-specific shRNA in transgenic pigs . Xenotransplantation 15, 36-45. a) Idee und Versuchsplanung b) Versuchsdurchführung c) Auswertung d) Verfassen des Manuskriptes Denner, J. Dieckhoff, B., Petersen, B., Kues, W.A., Dieckhoff, B. Dieckhoff, B., Kues, W.A., Niemann, H., Denner, J. Übersichtsartikel: Transgene Nutztiere, Stammzellen und DNA-Array Analysen Niemann, H., and Kues, W.A. 2003. Application of transgenesis in livestock for agriculture and biomedicine. Animal Reproduction Science 79, 291-317. a) Idee b) Umsetzung c) Verfassen des Manuskriptes Niemann, H., Kues, W.A. Niemann, H., Kues, W.A. Niemann, H., Kues, W.A. Kues, W.A. and Niemann, H. 2004. The contribution of farm animals to human health. Trends in Biotechnology 22, 286-294. a) Idee b) Umsetzung c) Verfassen des Manuskriptes Kues, W.A., Niemann, H. Kues, W.A., Niemann, H. Kues, W.A., Niemann, H. Kues, W.A., Carnwath, J.W., Niemann, H. 2005b. From fibroblasts and stem cells: Implications for cell therapies and somatic cloning. Reproduction, Fertility and Development 17, 125-134. a) Idee b) Umsetzung c) Verfassen des Manuskriptes Kues, W.A., Niemann, H. Kues, W.A., Niemann, H. Kues, W.A., Carnwath, J.W., Niemann, H. 57 Niemann H, Kues W, Carnwath JW. 2005. Transgenic farm animals: present and future. OIE Scientific and Technical Reviews (Rev. Sci. Tech. Off. Int. Epiz.) 24, 284-298. a) Idee b) Umsetzung c) Verfassen des Manuskriptes Niemann, H. Kues, W.A., Niemann, H. Niemann H, Kues W, Carnwath JW Niemann, H, Kues, W.A. 2007. Transgenic farm animals – an update. Reproduction, Fertility and Development 19, 762-770. a) Idee b) Umsetzung c) Verfassen des Manuskriptes Kues, W.A., Niemann, H Kues, W.A., Niemann, H. Kues, W.A., Niemann, H. Niemann, H., Carnwath, J.W., Kues, W.A. 2007. Application of array technology to mammalian embryos. Theriogenology 68S, S165-S177. a) Idee b) Umsetzung c) Verfassen des Manuskriptes Niemann, H. Kues, W.A., Niemann, H. Kues, W.A., Carnwath, J.W., Niemann, H. Kues, W.A., Rath, D., Niemann, H. 2008. Reproductive biotechnology goes genomics. CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources 3, No. 036, 1-18. a) Idee b) Umsetzung c) Verfassen des Manuskriptes Kues, W.A., Niemann, H. Kues, W.A., Niemann, H. Kues, W.A., Rath, D., Niemann, H. 58 9. Abkürzungsverzeichnis ACTB AdoMet AVR BAC Bp BSE CpG CMV DNA-MT EMEA ES Fo F1…n FACS FDA G0, G1 GDF8 GFP GH GMP FSSC hCD59 hDAF IRES 5mCpG mRNA NTA Oct4/OCT4 OG2 PA pCMV PERV PRNP ptTA RCA RISC RNAi siRNA shRNA SNP SPF STE SV40 tetO tet-off tet-on tg tTA wt ß-Actin-Gen S-Adenosylmethionin akute vasculäre Abstoßung bakterielles artifizielles Chromosom Basenpaar bovine spongiforme Enzephalitis Cytosin-Guanin-Dinukleotid Cytomegalovirus DNA-Methyltransferase European Medicine Agency embryonale Stammzelle Founder, transgenes Tier, das zur Gründung einer Linie genutzt wird erste Generation einer transgenen Linie, …nte Generation fluorescence activated cell sorting Food and Drug Agency Zellzyklusphase G0 (Ruhephase), G1 (gap1 der Interphase) Myostatin-Gen green fluorescent protein Wachstumshormon good manufacturing practices fetal somatic stem cell humaner Komplementregulator CD59 humaner Komplementregulator DAF, decay accelerating factor interne Ribosomenbindungsstelle 5-Methyl-CpG messenger RNA bicistronische Kassette mit tTA als zweitem Cistron octamer binding transcription factor 4 Oct4-GFP transgene Mauslinie 2 Polyadenylierungssequenz Promoter von CMV porcine endogene Retroviren Prionprotein-Gen tetracyclin-sensitiver Promoter, wird durch Bindung von tTA induziert regulator of complement activation, Komplementregulator RNA induced silencing complex RNA-Interferenz short interfering RNA short hairpin RNA Einzelbasenpolymorphismus spezifisch Pathogen-frei swine testis epithelial cell line Simian Virus 40 Tetracyclin-Operator konditionale Genexpression, tTA wird durch Tetracyclin inaktiviert konditionale Genexpression, mutierter tTA wird durch Tetracyclin aktiviert transgen; transgenes Tier tetracycline dependent transactivator wildtype; nicht-transgenes Tier 59 10. Anhang: Nachdruck der eigenen Publikationen In chronologischer Abfolge 60 Animal Reproduction Science 79 (2003) 291–317 Application of transgenesis in livestock for agriculture and biomedicine Heiner Niemann∗ , Wilfried A. Kues Department of Biotechnology, Institut für Tierzucht Mariensee, FAL, 31535 Neustadt, Germany Received 13 November 2002; received in revised form 27 January 2003; accepted 3 February 2003 Abstract Microinjection of foreign DNA into pronuclei of a fertilized oocyte has predominantly been used for the generation of transgenic livestock. This technology works reliably, but is inefficient and results in random integration and variable expression patterns in the transgenic offspring. Nevertheless, remarkable achievements have been made with this technology. By targeting expression to the mammary gland, numerous heterologous recombinant human proteins have been produced in large amounts which could be purified from milk of transgenic goats, sheep, cattle and rabbit. Products such as human anti-thrombin III, ␣-anti-trypsin and tissue plasminogen activator are currently in advanced clinical trials and are expected to be on the market within the next few years. Transgenic pigs that express human complement regulating proteins have been tested in their ability to serve as donors in human organ transplantation (i.e. xenotransplantation). In vitro and in vivo data convincingly show that the hyperacute rejection response can be overcome in a clinically acceptable manner by successful employing this strategy. It is anticipated that transgenic pigs will be available as donors for functional xenografts within a few years. Similarly, pigs may serve as donors for a variety of xenogenic cells and tissues. The recent developments in nuclear transfer and its merger with the growing genomic data allow a targeted and regulatable transgenic production. Systems for efficient homologous recombination in somatic cells are being developed and the adaptation of sophisticated molecular tools, already explored in mice, for transgenic livestock production is underway. The availability of these technologies are essential to maintain “genetic security” and to ensure absence of unwanted side effects. © 2003 Elsevier B.V. All rights reserved. Keywords: Nuclear transfer; Pronuclear DNA injection; Xenotransplantation; Recombinant proteins; Gene targeting ∗ Corresponding author. Tel.: +49-5034-871-148; fax: +49-5034-871-101. E-mail address: [email protected] (H. Niemann). 0378-4320/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-4320(03)00169-6 292 H. Niemann, W.A. Kues / Animal Reproduction Science 79 (2003) 291–317 1. Introduction: animal breeding and biotechnology Breeding of livestock has a long standing and very successful tradition. It began with domestication by which man habituated animals to live in his proximity. Using the available methodology man has propagated useful populations of animals. The selection was predominantly done on the basis of the phenotype and specific traits. A scientifically based animal breeding has existed for approximately 50 years on the basis of the increasing knowledge in population genetics and statistics. From the early beginning, this approach incorporated biotechnological procedures for which artificial insemination (AI) is the preeminent example. In cattle AI is employed in all countries with advanced breeding programs. Recently, significant increases in AI-application are also observed in swine where currently approximately 50% of the sows are artificially inseminated. This technology allows for a more efficient exploitation of the genetic potential of valuable sires and their propagation in a given population, frequently on a truly international scale. In the 1980s, embryo transfer technology has been transferred from an experimental stage to commercial application. This technology allowed for the first time a better exploitation of the genetic potential of the female in livestock breeding. As of today, >530,000 bovine embryos are transferred annually worldwide of which half are used upon freezing and thawing. In the other livestock species only very few embryos are transferred on a yearly basis (Thibier, 2001). The efforts of animal breeders and the application of artificial insemination, embryo transfer and further biotechnological procedures have resulted in the well known and remarkable increases in performances of livestock production. However, four major limitations have to be considered: 1. The genetic progress reaches only 1–3% per year and is thus relatively slow. 2. It is hardly or not possible to separate desired traits from non-desired traits. 3. A targeted transfer of genetic information between different species has not been possible. 4. Difficult adaptation to local conditions. Biotechnological procedures which have been under development during the past 20 years opened the perspectives to overcome the limitations listed above. Today, the term “biotech- Fig. 1. Generation of transgenic pigs for xenotransplantation. (A) Minigene construct for microinjection, PCMV : cytomegalovirus immediate early promoter, hCD59: human CD59 (regulator of complement) cDNA, PA: polyadenylation site, PvuI and AspI: flanking restriction enzyme sites. (B) DNA microinjection into one pronucleus of a porcine zygote. Prior to microinjection the zygote was centrifuged to separate the dark lipids from the cytoplasmatic fraction to allow optic identification of the pronuclei. (C) Transgenic founder animal identified by Southern blotting of an ear sample. (D) Organ-specific expression of hCD59 protein in an F1-offspring animal determined by Western blotting with a specific monoclonal antibody. (E) Cell surface expression of hCD59 in porcine primary cells as determined by flow cytometry. Grey shadowed curves demonstrated presence of hCD59 by usage of a monoclonal antibody, white curves indicate background values by usage of an isotype matched control antibody. (F) Functional expression of human CD59 on transgenic porcine cells as demonstrated by cytotoxicity assay. Porcine cells were incubated with heat treated human serum and a dilution series of human complement. Specific lysis of porcine cells were measured by chromium release. (G) Ex vivo perfusion of porcine kidneys from F1-animals with human blood. Nearly all transgenic porcine kidneys could be perfused for 4 h, whereas non-transgenic control kidneys failed soon after onset of perfusion due to hyperacute rejection. Mean survival times were 207.5 min for transgenic and 57.5 min for wildtype kidneys (P < 0.005) (Data from Niemann et al., 2001). H. Niemann, W.A. Kues / Animal Reproduction Science 79 (2003) 291–317 293 nology in livestock” comprises an arsenal of reproductive-biological and molecularbiological procedures. Reproductive biology includes: artificial insemination (AI), estrous synchronization, regulation of parturition, embryo transfer (ET), cryopreservation of gametes and embryos, sexing of sperm and embryos, in vitro production of embryos (IVP, including: in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC)), embryo bisection, nuclear transfer (NT, cloning), microinjection technology (DNA, RNA, 294 H. Niemann, W.A. Kues / Animal Reproduction Science 79 (2003) 291–317 Antisense). Molecular genetics comprise: genome analysis (sequencing, mapping, polymorphisms), molecular diagnostics (genetic defects (MHS, malignant hyperthermia syndrome; Dumps, deficiency of uridine monophosphate synthetase; Blad, bovine leucocyte adhesion deficiency), genetic descent, genetic diversity), functional genomics (expression patterns, interactions of genes), transgenics (additive gene transfer, knockout). Livestock biotechnology has an interdisciplinary character and comprises elements from anatomy, gynecology and obstetrics, endocrinology and physiology, andrology, ultrasoundtechnology, biochemistry, cell biology and molecular biology. The further development and refinement of bio- and gene technology with the goal to achieve commercial exploitation is considered to be an important tool to cope with future challenges in livestock production. A prominent example for biotechnological procedures with significant effects in animal production is the transgenic technology. Gene transfer is defined as the introduction of a protein coding DNA fragment into the host genome with the goal that the foreign DNA contributes to the protein synthesis of the host organism, e.g. the transgenic animal. Usually gene transfer involves gene constructs which are artificially combined DNA fragments consisting of regulatory and protein coding sequences. DNA microinjection and nuclear transfer are the two feasible technical approaches to generate transgenic livestock. Microinjection involves injection of several thousands of DNA copies into pronuclei of zygotes; zygotes are transferred into recipients and offspring is screened for integration and expression of the foreign DNA (Fig. 1A–D). Although this procedure is reliable, it is inefficient (1–4% transgenic offspring/transferred microinjected zygotes), results in random integration into the host genome and variable expression due to position effects (Pursel and Rexroad, 1993; Wall, 1996). In addition, it is time-consuming and requires substantial intellectual, financial and material resources (Seidel, 1993). Attributed to the enormous amounts of resources needed for transgenic livestock production, the costs for one expressing transgenic animal are extraordinary high. It has been calculated that one expressing transgenic mouse requires average expenses of 121 US$, whereas one expressing transgenic pig would amount to 25,000 US$, one transgenic sheep 60,000 US$ and one transgenic cow 546,000 US$ (Wall et al., 1992). Transgenic production in cattle can only be practical through in vitro production of embryos. From a total of more than 36,500 microinjected zygotes ≈2300 developed to blastocysts, upon transfer 28% resulted in pregnancy and 18 transgenic calves could be identified. To improve efficiency of the procedure the embryos were biopsied and analyzed by PCR for the presence of the transgene (Eyestone, 1999). The early detection of transgenesis in preimplantation embryos has been shown to be feasible, however, efficiency is limited due to an early incidence of mosaicism (Lemme et al., 1994). The propagation of the transgenic trait in a given cattle population can be accomplished through in vitro production techniques by using semen from a transgenic bull for in vitro fertilization and collecting oocytes by means of ultrasound-guided follicular aspiration from transgenic female founder animals and their subsequent use in IVF (Eyestone, 1999). Details of the microinjection technology and the potential applications of transgenic livestock have been extensively reviewed (Rexroad, 1992; Wall et al., 1992; Pursel and Rexroad, 1993; Niemann et al., 1994, 2002a; Wall, 1996; Murray, 1999). Despite the inherent limitations, microinjection has allowed commercial exploitation of transgenic technology with animals for biomedical purposes and even for few agricultural traits. Substantial progress in livestock transgenesis has been made through application of H. Niemann, W.A. Kues / Animal Reproduction Science 79 (2003) 291–317 295 somatic nuclear transfer. It is anticipated that the merger of nuclear transfer with molecular tools, such as targeted genetic modification and conditional gene expression already explored in mice, will provide another boost to livestock transgenesis (Niemann and Kues, 2000). 2. Current status of animal transgenesis 2.1. Transgenic animals with agricultural traits An Australian group has generated transgenic pigs bearing an hMt-pGH construct that can tightly be regulated by zinc feeding. The transgenic animals show significant improvements in economically important traits such as growth rate, feed conversion and bodyfat–muscle ratio (Nottle et al., 1997, 1999). Transgenic sheep carrying a keratin-IGF-I construct show expression in the skin and the clear fleece was about 6.2% greater in transgenic versus non-transgenic animals (Damak et al., 1996a,b). In both projects no adverse effects of the transgene on health or reproduction were observed. Another interesting application could be enhanced disease resistance (Müller and Brem, 1991). A mouse model in which recombinant monoclonal antibodies, which neutralize the transmissable gastroenteritis virus (TGV), are secreted into milk, provided passive protection against gastroenteric infections to the pups (Castilla et al., 1998). The verification of this model in pigs is promising. Recently, transgenic pigs expressing a bacterial phytase gene under the transcriptional control of a salivary gland-specific promoter were shown to have improved phosphate uptake (Golovan et al., 2001). The inserted phytase gene was almost exclusively expressed in the salivary gland and enabled the pigs to digest phosphorus in phytate, which could then be metabolized by the intestine. These animals require significantly fewer inorganic phosphate supplements, release substantially reduced phosphorus levels in manure and thus reduce environmental pollution. By transgenic expression of bovine alpha-lactalbumin in the mammary gland of sows, lactation performance was improved with respect to milk composition, milk yields and piglet growth (Wheeler et al., 2001). The increased survival of piglets at weaning provides significant benefits to animal welfare and the producer. 2.2. Transgenic animals in biomedicine 2.2.1. Gene pharming By targeting expression to the mammary gland via the use of mammary gland-specific promoter elements, large amounts of numerous heterologous recombinant proteins have been produced. In the bovine mammary gland of transgenic cows at least two different pharmaceutical proteins have been produced, in the caprine mammary gland five proteins, in the ovine system four proteins, in the pig two and in transgenic rabbits seven proteins (Rudolph, 1999). These could be purified from milk of transgenic rabbits, sheep, goats and cattle. The biological activity of the recombinant proteins was assessed and the therapeutic effects have been characterized (Rudolph, 1999; Meade et al., 1999). Human lactoferrin has recently been reported to be produced in large amounts in the mammary gland of transgenic cows (van Berkel et al., 2002). Products such as anti-thrombin III (ATIII), ␣-anti-trypsin 296 H. Niemann, W.A. Kues / Animal Reproduction Science 79 (2003) 291–317 Table 1 Status of selected recombinant pharmaceutical proteins produced in the mammary gland of transgenic livestock with regard to clinical testing/registration Donor species Product Clinical trial Treatment of Expected on market Company Sheep ␣-AT Phase II/III 2006 PPL/GTC Goat Goat Rabbit TPA AT III ␣-Glucosidasea Phase II/III Phase III Phase II/III ␣-AT-deficiency; cystic fibrosis Coronary clots Heparin-resistance Pompe’s disease 2006 2004 2004 GTC GTC Pharming a Orphan drug registration. (␣-AT) or tissue plasminogen activator (tPA) are currently in advanced clinical trials and are expected to be on the market within the next few years (Ziomek, 1998; Rudolph, 1999) (Table 1). On the basis of average expression levels, daily milk volumes and purification efficiency, 5400 cows would be needed to produce the 100,000 kg human serum albumin (HSA) that are required per year worldwide, 4500 sheep for the production of 5000 kg ␣-AT, 100 goats for 100 kg of monoclonal antibodies, 75 goats for the 75 kg ATIII and two pigs to produce 2 kg human clotting factor IX (Rudolph, 1999). Although a variety of proteins has been produced in the mammary gland of transgenic animals, not every protein can obviously be expressed at the desired high amounts. Erythropoietin (EPO) could not be expressed in the mammary gland of transgenic cattle (Hyttinen et al., 1994). We have shown that human clotting factor VIII cDNA constructs can be expressed in the mammary gland of transgenic mice, rabbit and sheep. However, the recovery rates of hFVIII protein were low and dependent on the donor, storage temperature and dilution of milk samples. hFVIII was rapidly sequestered in ovine milk (Halter et al., 1993; Espanion et al., 1997; Niemann et al., 1999; Hiripi et al., 2003). These latter results show that the technology needs further improvements to achieve high level expression of extraordinary large and complex regulated genes, such as hFVIII, although higher levels of hFVIII were reported in transgenic swine (Paleyanda et al., 1997). 2.2.2. Xenotransplantation 2.2.2.1. Transplantation of solid organs. Approximately 250,000 people are currently only living because of transplantation of an appropriate human organ (e.g. allotransplantation). In most cases no alternative therapeutic treatment was available and the recipients would have died without the organ transplantation. However, the enormous progress in organ transplantation technology which today is the basis for a normal life of thousands of patients has led to an acute shortage of appropriate organs, while the willingness to donate organs has remained unchanged or is even slightly reduced. Estimations in the USA have revealed that approximately 45,000 people, younger than 65 years need a heart transplant whereas only 2000 human hearts are transplanted annually (Michler, 1996). In the USA, more than 74,000 people are awaiting organ transplants and a new person is added to the waiting list every 14 min. Only 21,000 patients received a transplant in the year 2000 (Petit-Zeman, 2001). In Germany approximately 2400 kidneys, 730 livers, 540 hearts and 180 pancreas H. Niemann, W.A. Kues / Animal Reproduction Science 79 (2003) 291–317 297 are transplanted annually. However, the demand is twice as high as these figures. This has led to the sad and ethically challenging situation that several thousand patients die every year who could have survived if appropriate organs would have been available. To close this growing gap between demand and availability of appropriate organs, xenotransplantation (=the transplantation of organs between discordant species e.g. from animals to human) is considered as the solution of choice (Bach, 1998; Platt and Lin, 1998). The pig seems to be the optimal donor animal because • • • • • • the organs have a similar size as human organs, porcine anatomy and physiology are not too different from those in humans, pigs have short reproduction cycles and large litters, pigs grow rapidly, maintenance is possible at high hygienic standards at relatively low costs, pigs are a domesticated species. The process of evaluating transgenic pigs as potential donors for xenotransplants involves a variety of complex steps and is extremely time-, labor- and resource-intensive. Essential prerequisites for a successful xenotransplantation are: 1. Prevention of transmission of zoonoses from the donor animal to the human recipient. This aspect gained particular significance since a few years ago it was shown that porcine endogenous retroviruses (PERV) can be produced by porcine cell lines and can even infect human cell lines (Patience et al., 1997). However, until today no infection has been found in patients that had received various forms of living porcine tissues (e.g. islet cells, insulin, skin, extracorporal liver) for up to 12 years (Paradis et al., 1999). Recent intensive research revealed that PERV do not present a noticeable risk for recipients of xenotransplantation provided all necessary precautions are made (Patience et al., 1998; Dinsmore et al., 2000; Switzer et al., 2001; Martin et al., 2002). In addition, a strain of miniature pigs has been identified which does not produce infective PERU (Oldmixon et al., 2002). 2. Compatibility of the donor organs in anatomy and physiology with the human organ system, e.g. lifespan differences, growth rate, expected body weight. 3. Overcoming of the immunological rejection of the transplanted organ. The immunological hurdles are as follows (White, 1996a): (a) Hyperacute rejection response (HAR) occurs within seconds or minutes. In the case of a discordant organ, e.g. from pig to human, naturally occurring antibodies react with antigenic structures on the surface of the porcine organ and induce HAR by activating the complement cascade which is achieved via the antigen–antibody complex. Ultimately, this results in the formation of the membrane attack complex (MAC). However, the complement cascade can be shut down at various points by expression of regulatory genes which prevent the formation of MAC. Well known regulators of the complement cascade are CD55 (=decay accelerating factor, DAF), CD46 (=membrane cofactor protein, MCP) or CD59. MAC disrupts the endothelial cell layer of the blood vessels which leads to lysis, thrombosis, loss of vascular integrity and ultimately to rejection of the transplanted organ. (b) Acute vascular rejection (AVR) occurs within days. Induced xenoreactive antibodies are thought to be responsible for AVR. The endothelial cells of the graft’s 298 H. Niemann, W.A. Kues / Animal Reproduction Science 79 (2003) 291–317 microvasculature lose their anti-thrombic properties, attract leucocytes, monocytes and platelets leading to anemia and organ failure. (c) Cellular rejection occurs within weeks after transplantation. In this process the blood vessels of the transplanted organ are damaged by T-cells which invade the intercellular spaces and destroy the organ. This rejection is observed after allotransplantation and normally is suppressed by administration of immunosuppressive drugs. These have to be taken by the recipients for the rest of their lives. (d) Chronic rejection is a complex immunological process resulting in the rejection of the transplanted organ after several years. This process is slow and progressive and its etiology is largely unknown. The only remaining therapeutic option is another transplantation. When employing a discordant donor species such as the pig, overcoming the HAR is the preeminent goal. This cannot be achieved by administering high doses of an appropriate immunosuppressive drug as these do not affect the complement regulated rejection process. The most promising strategy to overcome the HAR is the synthesis of human complement regulatory proteins in transgenic pigs (Cozzi and White, 1995; White, 1996b; Bach, 1998; Platt and Lin, 1998). Following transplantation, the porcine organ would produce the complement regulatory protein and can thus prevent the complement attack of the recipient. Pigs transgenic for DAF have been generated and their hearts have been transplanted either heterotopically, e.g. in addition to the recipient’s own organ or orthotopically (=life supportive) into non-human primates. Upon heterotopic transplantation, the average survival of the recipients reached a maximum of 40–90 days, whereas the non-transgenic control organs were destroyed within a few minutes. The primates had to be treated with high doses of immunosuppressive drugs to maintain survival of the xenotransplant. Following moderate doses of immunosuppression survival rates of 20–25 days could be obtained (Bach et al., 1997; Cozzi et al., 2000). Employing the genomic clone of hCD46 (MCP), transgenic pigs showed a similar expression pattern for the transgene as found for the endogenous gene of the patients. Survival of a hCD46 porcine heart upon transplantation to baboons exceeded 23 days (Diamond et al., 2001). Similarly, transgenic expression of hCD59 was compatible with an extended survival of porcine hearts following transfer into primates (Fodor et al., 1994). Transplantation of hDAF-transgenic porcine kidneys was compatible with an extended survival of the recipients. The physiological function of the kidneys was maintained for up to 3–4 weeks (Zaidi et al., 1998) (Table 2). These data show that HAR can be overcome in a clinically acceptable manner by successful employing this strategy (Bach, 1998). Prior to primate experiments, extremely labor and time-consuming research is required to identify those transgenic animals with the most promising expression pattern of the transgene. Four research groups with strong links to the pharmaceutical industry have reported the generation of transgenic pigs with expression of human complement regulators. The screening of transgenic lines with suitable expression profiles is still inefficient. Previously, only one out of 30 lines showed an suitable expression pattern for xenotransplantation (Cozzi et al., 2000). A more efficient selection of transgenic pigs with an optimized expression pattern in two out of five tested lines was provided by employing the CMV promoter (Niemann et al., 2001). They provide a useful tool for the study of basic immunological H. Niemann, W.A. Kues / Animal Reproduction Science 79 (2003) 291–317 299 Table 2 Success rates of RCA-transgenic porcine organs upon transplantation to primate recipients RCA Organ/kind of transplant Recipient Immunosuppression Survival hDAF Heart/heterotopic Heterotopic Orthotopic Heterotopic Kidney/orthotopic Cynomologus Cynomologus Cynomologus Cynomologus Cynomologus +++a ++b +++ +c ++ ∼60 days ∼90 days ∼10 days ∼21 days ∼13 days (maximum 35 days) hCD59 Heart, heterotopic Baboon ++ ∼30 h hCD46 Heart, heterotopic Baboon ++ ∼23 days a Heavy immunosuppression. Moderate immunosuppression. c Weak immunosuppression. b questions in xenotransplantation. Transgenic pigs that show high expression of hCD59 predominantly in the heart, kidney and pancreas but also other target organs, were identified and transgenic lines established (Fig. 1D and E). Transgenic endothelial cells and fibroblasts were protected against complement mediated lysis (Fig. 1F). Perfusion studies using isolated porcine kidneys employing human blood revealed a significant protective effect against HAR (Fig. 1G). Orthotopic transplantation of a CMV-hCD59 transgenic porcine kidney into cynomologous monkey was compatible with extended survival of >20 days. Another promising strategy towards successful xenotransplantation is the knockout of the antigenic structures on the surface of the porcine organ. These structures are known as 1,3-␣-gal-epitopes and are produced from the gene for the 1,3-␣-galactosyltransferase. Recently, the generation of piglets in which one allele of the ␣-galactosyltransferase locus had been knocked out, was reported (Lai et al., 2002; Dai et al., 2002). These animals are now in breeding programs to obtain homozygous knockout animals. The usefulness of organs from these pigs for xenotransplantation is currently being tested. The birth of piglets with disruption of both allelic loci has recently been publicly announced. The strength of the cellular response to xenografts can be so great that it is unlikely to be fully controlled by immunosuppressive treatment and transgene expression. Further improvements of the success in xenotransplantation might arise from the possibility of inducing a permanent tolerance across xenogenic barriers (Greenstein and Sachs, 1997; Auchincloss and Sachs, 1998). A promising strategy for long-term graft acceptance seems to induce a permanent chimerism via intraportal injection of embryonic stem cell like structures (Fändrich et al., 2002). Although xenotransplantation poses numerous further challenges to research, it is expected that transgenic pigs will be available as organ donors within the next 5–10 years (Jones, 1996). Guidelines for the clinical application of porcine xenotransplants are currently being developed in several countries or are already available (USA). 2.2.2.2. Use of xenogenic cells and tissue. Another promising area of application for transgenic animals will be the supply of xenogenic cells and tissue. Several intractable diseases, disorders and injuries are associated with irreversible cell death and/or aberrant cellular func- 300 H. Niemann, W.A. Kues / Animal Reproduction Science 79 (2003) 291–317 tion. Despite numerous attempts, primary human cells cannot yet be expanded well enough in culture. In the future, human embryonic stem cells may serve as a source for specific differentiated cell types that can be used in cell therapy. Xenogenic cells, in particular from the pig, hold great promise with regard to a successful cell therapy for human patients (Edge et al., 1998). These cells provide several significant advantages over other approaches, such as implantation at the optimal therapeutic location (i.e. immunoprivileged sites such as the brain), possibility for manipulation prior to transplantation to enhance cell function, banking and cryopreservation, combination with different cell types in the same graft (Edge et al., 1998). There are already numerous examples for successful application of xenogenic cell therapy. Porcine islet cells have been transplanted to diabetic patients and were shown to be at least partially functional over a limited period of time (Groth et al., 1994). Porcine fetal neural cells were transplanted into the brain of patients suffering from Parkinson’s disease and Huntington’s disease (Deacon et al., 1997; Fink et al., 2000). In a single autopsied patient the graft survived for more than 7 months and the transplanted cells formed dopaminergic neurons and glial cells. Pig neurons extended axons from the graft site into the host brain (Deacon et al., 1997). Further examples for the potential use of porcine neural cells are stroke and focal epilepsy (Björklund, 1991). Human, fetal neuronal cells have also been employed as transplants into Parkinson’s and Huntington’s disease patients. The advantages of porcine neural cells over their human counterparts are the abundant availability and the option to introduce fail safe mechanisms via suicide genes. Olfactory ensheathing cells (OECs) or Schwann cells derived from hCD59 transgenic pigs promoted axonal regeneration in rat spinal cord lesion (Imaizumi et al., 2000). Thus, cells from genetically modified pigs may serve as therapeutic measure to restore electrophysiologically functional axons across the site of a spinal cord transsection. Xenogenic porcine cells may also be useful as novel therapy for liver diseases. Upon transplantation of porcine hepatocytes to Watanabe heritable hyperlipidemic (WHHL) rabbits (a model for familial hypercholesterolemia), the xenogenic cells migrated out of the vessels and integrated into the hepatic parenchyma. The integrated porcine hepatocytes provided functional LDL receptors and thus reduced cholesterol levels by 30–60% for at least 100 days (Gunsalus et al., 1997). A clone of bovine adrenocortical cells restored adrenal function upon transplantation to adrenalectomized SCID mice. This finding shows that functional endocrine tissue can be derived from a single somatic cell (Thomas et al., 1997). Bovine neuronal cells were collected from transgenic fetuses, transplanted into the brain of rats and resulted in significant improvements of symptoms of Parkinson’s disease (Zawada et al., 1998). Furthermore, xenotransplantation of retinal pigment epithelial cells holds promise to treat retinal diseases such as macular degeneration which is associated with photoreceptor losses. Porcine or bovine fetal cardiomyocytes or myoblasts may provide a therapeutic approach for the treatment of ischemic heart disease. Similarly, xenogenic porcine cells may be valuable for the repair of skin or cartilage damage (Edge et al., 1998). In light of the emergence of more efficient protocols for genetic modification of donor pigs and new powerful immunosuppressive drugs, one can expect xenogenic cell therapy to evolve as an important therapeutic option for the treatment of human diseases. The above description demonstrates that although the challenges to generate a transgenic animal with an optimized tailored expression pattern are enormous, within less than 20 years transgenic livestock have emerged that provide valuable contributions to human health. H. Niemann, W.A. Kues / Animal Reproduction Science 79 (2003) 291–317 301 With increasing knowledge about the genetic basis of agricultural traits and improvements in the technology to generate transgenic animals, numerous further useful commercial applications will be developed. 3. Improvements of transgenic technology 3.1. Inducible gene expression A major advancement in transgenic technology would be tight control of transgene expression. Control elements that are known to regulate the activity of transgenes are the metallothionein promoter (see Nottle et al., 1999), heat shock promoter, or steroid responsive elements. However, these have been used with limited success attributed to low induction levels and physiological effects of the inducer elements (Yarranton, 1992). Significant improvements of the temporal control of gene expression could be achieved by employing the antibiotic tetracycline system (Gossen et al., 1995). This involves a transcriptional transactivator which has been created by fusion of the VP16 activation domain with a mutant Tet repressor from Escherichia coli. This transactivator requires the presence of tetracycline or an analogue for DNA binding and transcriptional activation (Fig. 2). It has been shown that Fig. 2. Schematic drawing of Tet-on system. In the original system two independent transgene constructs were integrated in the genome. The transactivator gene is continuously transcribed and the inactive form of the transactivator is translated. By exogenous addition (feeding) of a tetracycline analogue the transactivator becomes activated, subsequently binds to the Tet-promoter and induces expression of the transgene. Removal of the compound leads to transcriptional silencing of the transgene. 302 H. Niemann, W.A. Kues / Animal Reproduction Science 79 (2003) 291–317 Fig. 3. Conditional transgene expression in porcine muscle. (A) Transgenic pigs carrying a Tet-off expression cassette show massive expression in skeletal muscle fibers. Muscle biopsies of a wildtype and a transgenic animal were stained with a specific antibody against the transgene. Arrows indicate a positive muscle fiber. (B) Conditional ablation of transgene expression. Expression levels of the transgene were determined in muscle biopsies before and after feeding with doxycycline. In total, four animals were treated with doxycyline and showed identical downregulation of expression. the presence of a tetracycline analogue led to a burst of expression in cell lines and even transgenic mice (Tet-on) (Gossen et al., 1995). This system can also be modified in a way that the presence of tetracycline suppresses expression of the target gene (Tet-off). In this system, tetracycline binds to the transactivator and shuts down expression of the transgene (Furth et al., 1994). The original technology requires two constructs, which make it unfeasible for application in livestock. However, it has been shown that fusion of both control elements into one construct allows efficient and tight control of gene expression in vivo (Schultze et al., 1996). In recent own work, the transgene expression in pigs, carrying a single Tet-off gene construct, could be regulated by doxycycline feeding (Fig. 3, unpublished data). Further studies will show the usefulness of this system for the conditional expression of transgenes suitable in xenotransplantation. 3.2. Artificial chromosomes: YACs, BACs and MACs Artificial chromosomes are DNA molecules of predictable structure which are assembled in vitro from defined constituents that are similar to natural chromosomes. The first artificial chromosomes have been constructed in yeast (Saccharomyces cerevisiae) (Fig. 4). They include centromeres, telomeres, and origins of replication as essential components (Brown et al., 2000). These yeast artificial chromosomes (YACs) can be introduced into cell lines (Strauss et al., 1993). They carry much larger amounts of DNA than usually can be employed in microinjection. Microinjection of a 450 kb genomic YAC harboring the H. Niemann, W.A. Kues / Animal Reproduction Science 79 (2003) 291–317 303 Fig. 4. Schematic comparison of yeast and mammalian artificial chromosomes used for the generation of transgenic animals. Injection of yeast or bacterial artificial chromosomes (YAC, BAC) into mammalian zygotes leads to a random integration, since their structural chromosomal elements are not functional in mammalian cells. In contrast, injection of mammalian artificial chromosomes results in the inheritance of these vectors as independent chromosomal elements. murine tyrosinase gene resulted in transgenic mice which showed position independent and copy number dependent expression of the transgene. Albinism was rescued in transgenic mice and rabbits (Schedl et al., 1992, 1993; Brem et al., 1996). A 210 kb YAC construct has been microinjected into rat pronuclei and ␣-lactoglobulin and human growth factor were expressed in the mammary gland of transgenic rats (Fujiwara et al., 1997, 1999). Artificial chromosomes can also be constructed in bacteria (BACs), which can be genetically modified easier and even allow homologous recombination. Transgenic mice were generated via pronuclear injection of BACs and germline transmission and proper expression of the transgene was achieved (Yang et al., 1997). However, up to now, transgenic livestock has not been 304 H. Niemann, W.A. Kues / Animal Reproduction Science 79 (2003) 291–317 reported upon transfer of a YAC construct. This may be attributed to the inherent problems of this technology, such as difficulties to isolate YAC DNA with sufficient purity and the inherent instability with a tendency for deleting regions from the insert (Monaco and Larin, 1994). Mammalian artificial chromosomes (MAC) have been engineered by employing endogenous chromosomal elements from YACs or extra chromosomal elements from viruses or BACs and P1 artificial chromosomes (PACs) (Vos, 1997). MACs with a size of 1–5 Mb were formed by a de novo mechanism and segregated like normal chromosomes upon introduction into cell lines (Ikeno et al., 1998). A human artificial chromosome (HAC) containing the entire sequences of the human immunoglobulin heavy and light chain loci has been introduced into bovine fibroblasts, which were then used in nuclear transfer. Transchromosomal offspring were obtained that expressed human immunoglobulin in their blood. This system could be a significant step forward in the production of therapeutic polyclonal antibodies (Kuroiwa et al., 2002). Satellite-DNA based artificial chromosomes (SATAC) are neochromosomes that are formed by de novo amplification of pericentric heterochromatin yielding chromosomes from 10 to 360 megabases. These can serve as chromosomal vectors for exogenous DNA (Perez et al., 2000). Transgenic mice have been generated by microinjection of SATACs into pronuclei of zygotes. The additional chromosome showed germline transmission over three generations (Co et al., 2000). Microinjection of SATACs was also compatible with the development of bovine embryos. Transgenic embryos could be identified by staining for the presence of a reporter gene and FISH detection of the extra chromosome (Co et al., 2000). Moreover, SATACs could be isolated and purified by flow cytometry due to their high AT content. However, all efforts to assemble MACs as functional vectors for foreign DNA have met with limited success. Mini-chromosomes are considered to overcome the problems with artificial chromosomes. In contrast to artificial chromosomes, mini-chromosomes possess a defined structure, can be engineered to harbor large fragments of DNA and can go through the vertebrate germline (Brown et al., 2000). Mini-chromosomes could play an important role in the development of transchromosomal animals as models of human genetic disorders such as trisomy 21 (Down’s syndrome) (Hernandez et al., 1999). Recently, mice have been engineered that contain certain fragments of human chromosomes with each of the immunoglobulin heavy chain and light chain gene. In these animals, the endogenous immunoglobulin genes were deleted and the mice produced humanized antibodies (Tomizuka et al., 2000). Further progress in this area will ultimately result in a variety of different vector systems which can be used for large scale transgenesis in experimental animals, agricultural livestock and crop plants. 4. Nuclear transfer and targeted genetic modification 4.1. Nuclear transfer technology: results and limitations As microinjection has a number of significant shortcomings mentioned above, research has focused on alternate methodologies for improving the generation of transgenic livestock. These include sperm mediated DNA transfer (Gandolfi, 1998; Squires, 1999; Chang et al., H. Niemann, W.A. Kues / Animal Reproduction Science 79 (2003) 291–317 305 2002; Lavitrano et al., 2002), the intracytoplasmic injection (ICSI) of sperm heads carrying foreign DNA (Perry et al., 1999, 2001), the use of retroviral vectors either by injection or infection of oocytes or embryos (Haskell and Bowen, 1995; Chan et al., 1998; Cabot et al., 2001) or the use of genetically modified donor cells from livestock in nuclear transfer (Schnieke et al., 1997; Cibelli et al., 1998a; Baguisi et al., 1999; Park et al., 2001). Further improvements may be derived from the adaptation of technologies that allow precise modifications of the murine genome. These include targeted chromosomal integration by site-specific DNA recombinases, such as Cre or FLP or homologous recombination that would enable generation of transgenic animals with a gain (knockin) or a loss of function (knockout) (Capecchi, 1989; Kilby et al., 1993). In light of the recent advances, somatic nuclear transfer holds the greatest promise for significant improvements in the generation of transgenic livestock. A major prerequisite is the availability of suitable primary cells or cell lines compatible with techniques for precise genetic modifications either for gain or loss of function. Another prerequisite is a significantly improved knowledge of gene sequences and organization of the livestock genome, which currently is lagging much behind that of mouse and human. In the latter, the putative 3 billion base pairs have been sequenced in the year 2001. Surprisingly, the human genome only contained approximately 30–35,000 genes (Baltimore, 2001). However, RNA editing and alternative splicing significantly augments the number of proteins synthesized from a gene. Whereas only 250 out of the 6000 genes in Saccharomyces cerevisiae contain introns, the vast majority of the estimated human genes are thought to contain these structures (Graveley, 2001; Modrek and Lee, 2001). In contrast, in livestock species only a minority of genes have been mapped and sequenced until now (Table 3). However, the technology developed during deciphering the human genome will improve and accelerate sequencing of genomes from other species (O’Brien et al., 1999). Even the currently limited genetic information in livestock species allows the application of cDNA array technologies and or high density DNA chips to obtain gene expression profiles of nearly any tissue of interest. Improvements of RNA isolation and unbiased amplification of tiny amounts of mRNA (picogram) enable to analyze even single embryos (Brambrink et al., 2002). Reports on the generation of transgenic livestock (Schnieke et al., 1997; Cibelli et al., 1998a; Park et al., 2001) via somatic nuclear transfer had raised great expectations about this elegant approach to improve the generation of transgenic livestock. Fetal fibroblasts were transfected in vitro, screened for transgene integration and then transferred into enucleated oocytes. After fusion of both components and activation of the reconstituted nuclear transfer complexes, blastocysts were transferred to synchronized recipients and gave rise to transgenic offspring. Compared with the microinjection procedure in which screening for transgenesis and optimal expression of the transgenes takes place at the level of the offspring, cloning by nuclear transfer can accelerate the time-consuming transgenic production by rapid screening in vitro and 100% transgenic offspring. The first commercial data of the use of nuclear transfer to generate transgenic cattle show the feasibility of this approach (Forsberg et al., 2001). Offspring from nuclear transfer have been born in all major livestock species cattle, sheep, goat and swine (Wilmut et al., 1997; Cibelli et al., 1998b; Baguisi et al., 1999; Polejaeva et al., 2000; Betthauser et al., 2000). A variety of different cell types of embryonic, fetal and somatic origin has been successfully employed as donors in nuclear transfer. However, the overall efficiency of nuclear transfer is low (Colman, 2000). Factors affecting 306 H. Niemann, W.A. Kues / Animal Reproduction Science 79 (2003) 291–317 Table 3 Whole genome sequencing Species No. annotated genes Milestones in genome sequencing and size of selected genomes Haemophilus influenca (bacteria) ∼1700 Saccaromyces cerevisae (fungi) ∼6000 Caenorhabditis elegans (nematodes) ∼18000 Drosophila melanogaster (insects) ∼14000 Arabidopsis thalia (plants) ∼25000 Homo sapiensa (mammals) ∼35000 Genome size (×106 bp) 1.7 12.0 97.0 137.0 125.0 3300.0 Sequencing completed 1995 1996 1998 2000 2000 2001 Diversity of genome sequencing approaches (10/2002) Archaea Bacteria Eukaryota Complete genomes of 16 species Complete genomes of 81 species complete genomes of 9 species. In addition the genomes of Homo sapiens, Mus musculus, Rattus norvegicus, Danio rerio and some crop plants are mapped, however gene annotation and/or sequencing of untranscribed regions is still in progress Species Chromosome number (haploid) No. mapped genes Humanb Mousec Cattlec Sheepc Pigc Horsec Dogc Chickenc 23 20 30 27 19 32 39 39 >30000 30000 ∼1000 ∼500 ∼600 <200 ∼250 >200 Genome size (×1000) 3.300 2.450 ∼3.500 ∼3.100 ∼2.400 ∼2.000 ∼2.700 ∼4.000 a Gene annotation still in progress. February 2001 sequencing completed. c Information for 2002. b the success of nuclear transfer are poorly defined and the average percentage of live offspring does not exceed 1–3% of the transferred reconstituted embryos (Wakayama et al., 1998; Wilmut et al., 2002). A better understanding of the underlying fundamental molecular and cellular processes, such as cell cycle compatibilities between recipient cytoplasm and donor nucleus (Campbell et al., 1996), cell cycle synchronization of the donor cells (Boquest et al., 1999; Kues et al., 2000), reprogramming and the relevance of differentiation versus totipotency is urgently needed. Upon serum deprivation or treatment with chemical cell cycle inhibitors, the majority of porcine donor cells was synchronized at the presumptive optimal cell cycle stage at G0 /G1 without compromising their viability (Kues et al., 2000, 2002; Anger et al., 2003). This contributes substantially to standardize the nuclear transfer procedures. In addition, methods have to be established that allow reliable determination H. Niemann, W.A. Kues / Animal Reproduction Science 79 (2003) 291–317 307 of the capacity of a given nuclear transfer embryo to develop into a normal offspring. The large offspring syndrome (LOS) including an increased peri- and postnatal mortality, is found in offspring, predominantly from ruminants, derived from nuclear transfer embryos (Wilmut et al., 1997; Young et al., 1998; Kato et al., 1998). These unwanted side effects need to be overcome prior to an eventual commercial exploitation. Aberrations of the well orchestrated pattern of gene expression are thought to be involved in the high incidence of LOS. A primary mechanism may be alterations in the methylation of genes, including those that are subject to imprinting (Young et al., 1998; Niemann and Wrenzycki, 2000; Niemann et al., 2002b). 4.2. Embryonic and adult cell lines The fundamental tools for loss-of-function transgenics in the mouse are the availability of embryonic stem cells (ES cells), homologous recombination and the high probability with which ES cells give rise to germline contribution after injection into host blastocysts. This provides a powerful approach to introduce specific genetic changes into the murine genome (Evans and Kaufman, 1981; Martin, 1981). The essential characteristics of ES cells include derivation from the preimplantation embryo (inner cell mass cells), undifferentiated and indefinitive proliferation in vitro and the developmental potential to differentiate into all cell types under appropriate conditions. Besides ES cells, embryonic germ (EG) and embryonic carcinoma (EC) cells have been established in the mouse model. EG cells are isolated from cultured primordial germ cells (PGC) (Matsui et al., 1992; Resnick et al., 1992) and share several characteristics with ES cells, including morphology, pluripotency, and the capacity for germline transmission. Similar to ES cells, EG cells express alkaline phosphatase and Oct-4 and can be aggregated to form embryoid bodies. The ultimate criterion for true totipotent stem cell lines is the contribution to the germline in chimeras or starting a new development upon nuclear transfer. ES or EG-like cell lines have been isolated from sheep, goat, pig and cattle (Wheeler, 1994; Anderson, 1999). Porcine and bovine cell lines were capable to contribute to chimera formation upon injection into appropriate host blastocysts (Wheeler, 1994; Shim et al., 1997; Piedrahita et al., 1998; Cibelli et al., 1998a). However, no germline transmission has been reported so far. True totipotent stem cell lines might require specific culture conditions, growth factor supplements and probably a specific genetic background, as only few mouse strains are suitable for ES cell line isolation (Hogan et al., 1994). Recent experiments in mice have revealed that adult cells from the hematopoetic lineage possess a greater plasticity than previously assumed (Jiang et al., 2002). Besides long-term proliferation in vitro, these mesenchymal derived cells express embryonic markers, such as Oct-4, rex and telomerase and can be differentiated into several cell types in vitro and in vivo. Upon injection into blastocysts, chimeric mice could be generated, which showed a high percentage of chimerism in nearly all organs. Similar cells were isolated from the hematopoetic system of rats and even humans, suggesting that mesenchymal stem cells could also be a useful source of research and application in livestock species. 308 H. Niemann, W.A. Kues / Animal Reproduction Science 79 (2003) 291–317 4.3. Homologous recombination and nucleus donor cell strains Homologous recombination in murine ES cells is the most straightforward approach to eliminate gene function and is therefore the preferred method to establish a null genotype. Several strategies for gene targeting in murine ES cells have been developed (Kühn et al., 1995; Mayford et al., 1997). More than 1000 knockout strains have been created via gene targeting in embryonic stem cells (mouse knockout and mutation database http://www.biomednet.com). The potential for a knockout technology in livestock production is highlighted by the discovery that several beef cattle breeds, like Belgian Blue and Piedmontese, are accidentally homozygous for the mutated myostatin gene, which is functionally inactive and could be referred to as a natural knockout (McPherron and Lee, 1997; Kambadur et al., 1997; Grobet et al., 1997). The similarity in phenotypes of myostatin mutated cattle and myostatin null mice (McPherron et al., 1997) is striking and suggests that myostatin could be a useful target for genetic modification in farm animals. Nuclear transfer techniques promise to circumvent the need for true totipotent cells for the generation of loss-of-function transgenic livestock. The future challenge for the production of transgenic livestock is the isolation and handling of primary cell cultures, either from somatic or embryonic origin (Schnieke et al., 1997; Cibelli et al., 1998b; Kues et al., 1998). These could be used for sophisticated genetic modifications, clonal selection and subsequently for nuclear transfer. Gene targeting in somatic cells of livestock has important applications in combination with nuclear transfer (Fig. 5). Recent progress in the generation of gene targeted livestock clearly shows the significant potential of this field. An efficient and reproducible targeting protocol in fetal fibroblasts by which the transgenic construct -lactoglobulin-␣-anti-trypsin was placed at the ovine ␣1 precollagen locus was described and the production of live sheep upon nuclear transfer was reported (McCreath et al., 2000). The recombinant ␣-AT was highly expressed in the mammary gland. However, the proportion of embryonic and fetal losses was significantly increased in pregnancies with targeted NT derived sheep and porcine embryos. The first piglets, carrying a knockout for one allele of the ␣-galactosyltransferase gene did not show gross abnormalities (Lai et al., 2002; Dai et al., 2002). By performing two rounds of targeting in cell culture, both alleles can be knocked out and time-consuming breeding will be avoided in future (Fig. 5). Exploiting the targeting technology would allow to produce large amounts of human serum albumin (HSA) when inserted at the BSA locus in transgenic cattle. A knockout of the PrP gene locus could render cattle non-susceptible to BSE. Disease models in large animals would be another promising application models for targeted transgenesis. The pig is obviously a good model for human eye disease (Petters et al., 1997; Theuring et al., 1997) and sheep could be a good model for cystic fibrosis. 4.4. Conditional mutagenesis and region-specific knockouts The above findings demonstrate the power of gene targeting, but they also point to a need for additional genetic techniques when precise genetic modifications are desired. Gene knockouts per se have no spatial or temporal restriction. As the targeted gene product H. Niemann, W.A. Kues / Animal Reproduction Science 79 (2003) 291–317 309 Fig. 5. Schematic drawing of gene targeting in livestock. Gene targeting of somatic primary cells by homologous recombination employing a promoterless targeting vector. Optionally, cell clones with the desired targeting event could be screened for loss of heterozygosity and subsequently be employed in nuclear transfer. is absent for the entire life of the animal in all cells, it is difficult to assign a phenotype to a specific knockout as a mutant organism may compensate for the loss of a gene product or the knockout may have complex, secondary effects, depending on the genetic background (Gerlai, 1996), or may even result in embryonic lethality. A powerful tool for the design of genetic switches and for accelerating the creation of genetically modified animals, is the Cre site-specific DNA recombinase of bacteriophage P1 (Sauer, 1998). A single 38 kDa Cre protein is required to catalyze recombination between two loxP recognition sites, which are 34 bp DNA sequences. Recombination can occur between directly repeated loxP sites on the same molecule to excise the intervening DNA sequence, irrespective of whether the recognition sites are located on a plasmid or an mammalian chromosome (Sauer and Henderson, 1988). The combination of tissue-specific promoter elements with Cre DNA recombinase, enables a gene to be knocked out of a restricted cell type or tissue (Orban et al., 1992; Gu et al., 1994). In principle, recombinase mediated recombination should allow gene replacement, e.g. the exchange of the reading frames of milk proteins with the sequences of genes encoding pharmaceutical proteins. 310 H. Niemann, W.A. Kues / Animal Reproduction Science 79 (2003) 291–317 5. Perspectives and outlook The merger of the recent advancements in the reproductive technologies with the tools of molecular biology opens the horizon for a completely new era in animal production. However, major prerequisites will be the continuous refinement of reproductive biotechnologies and a rapid increase of livestock genome mapping. This would permit exploitation the great potential of bio- and gene technology with regard to a diversified animal production. One attractive example could be dairy production (Bawden et al., 1994). Apart from conventional dairy products, it could be possible to produce fat-reduced or even fat-free milk via knockout of enzymes involved in lipid metabolism, to increase curd and cheese production by enhancing expression of the casein gene family in the mammary gland, to increase efficiency of the production for creamers and liquor by increasing the proportion of -casein in milk, to create “hypoallergic” milk by knockout of the -lactoglobulin gene, to generate lactose-free milk via knockout of ␣-lactalbumin which is the key molecule in milk sugar synthesis, to produce small infant milk in which human lactoferrin is abundantly available or to produce milk with a highly improved hygienic standard via an increased level of lysozyme or other anti-microbiological substances in the udder. Lactose free-milk could render dairy products ready for consumption by a large proportion of the world’s population who do not possess the active enzyme lactase in their intestine. In addition, calculations have revealed that reduction of the fat level in milk from the current level of 3.8–2% would permit an increase in the proportion of roughage in the feed from 55 to more than 80% and concomitantly to a significant reduction in the concentrated feed (Yom and Bremel, 1993). This would reduce the price and cost of animal nutrition. Such targeted production is also imaginable for other areas of animal production. However, one has to take into account that commercial application of this technology for agricultural traits will likely not be possible before the next decade. The biomedical area will see the first commercial application before the year 2010 (Table 4). On a broader scale, bio- and gene technology as mentioned above will be important tools to cope with the challenges in animal production in the years ahead. In light of the worldwide problems in the management of a proper environment and resources, biotechnology can contribute to quantitative and qualitative increases in food production, cost reduction, Table 4 Projections for field application of transgenic livestocka Year Biomedical traits Recombinant pharmaceutical proteins Xenogenic cells/tissue Xenotransplantation of solid organs <2004 >2005 >2008 Agricultural traits Dairy products Meat and meat products Wool products >2010 >2008 >2005 a Application will likely be different between USA and Europe because of the controversial debate on genetic engineering in Europe. H. Niemann, W.A. Kues / Animal Reproduction Science 79 (2003) 291–317 311 environmental protection, maintenance of genetic resources and improvements in animal welfare as well as the above mentioned diversified production. However, it should be kept in mind that putative biomedical applications like xenotransplantation or usage of transgenic farm animals for food production will require strict standards on “genetic security” and reliable and sensitive methods for the molecular characterization of the “products”. A major contribution towards this goal will come from DNA chips or arrays establishing “fingerprint” profiles at the transcriptional and/or protein level and allowing in depth insight into the proper function of a transgenic organism. Besides the technical problems, public acceptance of recombinant products, organs or food has to be taken into account. Additionally, welfare of the transgenic animals must be secured and pain or suffering due to the genetic modification could not be tolerated (Fox, 1988; Hughes et al., 1996). Acknowledgements The authors express their sincere gratitude to Christa Knöchelmann for the careful preparation of this manuscript. References Anderson, G.B., 1999. 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KUES,2 JIRI KLIMA,1 MANFRED MIELENZ,2 MICHAL KUBELKA,1 JAN MOTLIK,1 MILAN ESNER,3 PETR DVORAK,3 JOSEPH W. CARNWATH,2 AND HEINER NIEMANN2* 1 Institute of Animal Physiology and Genetics, Libechov, Czech Republic 2 Department of Biotechnology, Institute for Animal Science, Mariensee, Neustadt, Germany 3 Laboratory of Molecular Embryology, Mendel University, Brno, Czech Republic ABSTRACT Enzymes of the Polo-like kinase (Plk) family are active in the pathways controlling mitosis in several species. We have cloned cDNA fragments of the porcine homologues of Plk1, Plk2, and Plk3 employing fetal fibroblasts as source. All three partial cDNAs showed high sequence homology with their mouse and human counterparts and contained the Polo box, a domain characteristic for all Polo kinases. The expression levels of Plk1 mRNA at various points of the cell cycle in synchronized porcine fetal fibroblasts were analyzed by both RT-PCR and the ribonuclease protection assay. Plk1 mRNA was barely detectable in G0 and G1, increased during S phase and peaked after the G2/M transition. A monoclonal antibody was generated against an in vitro expressed porcine Plk1-protein fragment and used to detect changes in Plk1 expression at the protein level. Plk1 protein was first detected by immunoblotting at the beginning of S phase and was highest after the G2/M transition. In summary, the Plk1 expression pattern in the pig is similar to that reported for other species. The absence of Plk1 mRNA and protein appears to be a good marker for G0/G1 and thus for the selection of donor cells for nuclear transfer based somatic cloning. Mol. Reprod. Dev. 65: 245–253, 2003. ß 2003 Wiley-Liss, Inc. Key Words: Plk1; serum deprivation; cell cycle; butyrolactonuI; aphidicolin INTRODUCTION Polo kinase was first identified in yeast and Drosophila (Llamazares et al., 1991). The homologous molecule, Polo-like kinase (Plk), was subsequently isolated from higher vertebrates including, mouse (Clay et al., 1993), human (Lake and Jelinek, 1993), and Xenopus (Kumagai and Dunphy, 1996). All members of the Polo kinase family contain two copies of a unique C-terminal sequence, the ‘‘Polo box,’’ thought to be responsible for the intracellular localization of the enzyme (Clay et al., 1997; Lee et al., 1998). Two additional kinases belonging to the Polo family but unique to vertebrates are called Plk2 and Plk3 (Kauselmann et al., 1999; Holtrich et al., 2000; Hudson et al., 2001; Nigg, 2001). Since there are no homologues of Plk2 and Plk3 in lower eukaryotes, ß 2003 WILEY-LISS, INC. detailed knowledge about the role of the Polo kinases relates to Plk1. Plk1 is abundant in proliferating cells and tissues including fetal tissues and certain types of human cancer (Yuan et al., 1997; Wolf et al., 1997, 2000). In cultured mouse cells, Plk1 mRNA is first detectable at G1/S transition, with a peak at G2/M transition (Lake and Jelinek, 1993; Uchiumi et al., 1997). Several studies have demonstrated that Plk1 is critically involved in cell cycle related activities. The kinase activity of Plk1 is highest at G2/M and when it is phosphorylated during M phase and timing of this activation is coincident with activation of MPF (Golsteyn et al., 1995; Hamanaka et al., 1995; Mundt et al., 1997). Indeed, it has been suggested that activated Plk1 is involved in activation of MPF and thus on mitotic entry (Roshak et al., 2000). Plk1 activity regulates maturation of the spindle; is involved in checkpoint monitoring of the integrity of replicated DNA (Smits et al., 2000), and regulates substrate specificity of the anaphase-promoting complex (APC) (reviewed by Glover et al., 1996, 1998; Golsteyn et al., 1996; Nigg, 1998, 2001). In late mitosis, Plk1 appears to be involved in the control of cyclin B proteolysis, thus initiating processes resulting in the exit from mitosis (Abrieu et al., 1998; Descombes and Nigg, 1998; Ferris et al., 1998; Kotani et al., 1998; Qian et al., 1998; 1999; Karaiskou et al., 1999). In the present study, we have cloned the porcine Plks 1, 2, and 3 and compared the mRNA expression levels of these genes in synchronized murine and porcine fetal fibroblasts throughout the cell cycle. In addition, a monoclonal antibody against Plk1 was generated and characterized. Expression of Plks in porcine cells had not been reported previously. The data indicate that Plk1 mRNA shows the most dramatic changes during Grant sponsor: FIRCA; Grant number: R03-TW-05530-01; Grant sponsor: German Ministry for Education and Research (BMBF); Grant sponsor: Ministry of the Education Youth and Sport of the Czech Republic; Grant number: LN00A065. *Correspondence to: Prof., Dr. Heiner Niemann, Department of Biotechnology, Institute for Animal Science, Mariensee, D-31535 Neustadt, Germany. E-mail: [email protected] Received 19 August 2002; Accepted 13 January 2003 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mrd.10289 246 M. ANGER ET AL. cell cycle in porcine fetal fibroblasts. We further tested the hypothesis that absence of Plk1 mRNA and protein in nonproliferating cells and tissues (Kues et al., 2000), i.e., cells in the G0 and G1 phase of the cell cycle, provides a suitable marker to identify populations of growth-arrested cells. To do this, expression levels of Plk1 mRNA and protein were measured after fetal calf serum (FCS) stimulation of serum deprived cells for defined time intervals as well as in cells blocked by either aphidicolin or aphidocolin and butyrolactone I treatment. Determining Plk1 expression to define the cell cycle stage is of great interest because these cells are frequently employed as nucleus donor cells in somatic cloning experiments (Onishi et al., 2000; Polejaeva et al., 2000) and it had been suggested that it is beneficial to block donor cells in G0 by serum deprivation (Wilmut and Campbell, 1998; Lee and Piedrahita, 2002). MATERIALS AND METHODS Cell Culture, Cell-Cycle Synchronization, and FACS Analysis STO and HeLa cells were purchased from ATTCC (American Type Culture Collection, Manassas VA), bovine skin fibroblasts were a kind gift from Dr. Jiri Kanka, mouse fetal fibroblasts were prepared according to standard protocols (Hogan et al., 1994). Cells were thawed and grown to 80% confluency in Dulbecco’s Modified Eagle Medium (DMEM) plus 10% FCS (Sigma Chemical Co., St. Louis, MO). Mouse fetal fibroblasts were synchronized in G0/G1 by reduction of serum content to 0.2% for 3 days followed by stimulation with 10% FCS 1, 11, 16, and 21 hr before harvesting. Porcine primary fibroblasts were prepared as recently described (Kues et al., 2000). All experiments were performed from the same batch of fetal fibroblasts harvested after the 2nd passage, frozen and stored in liquid nitrogen until use. For each series of experiments, fibroblasts were thawed and grown to 80% confluency in DMEM plus 10% FCS (Sigma Chemical Co.). To obtain cell-cycle expression profiles for Plk1, Plk2, and Plk3 mRNAs, cells were synchronized by deprivation of FCS (0.2%) in culture media for 3 days followed by stimulation with 10% FCS for 1, 4, 18, 21 hr. Nocodazole (Sigma Chemical Co.) was used to obtain M phase synchronized cells. Therefore, cells stimulated for 16 hr with 10% FCS were synchronized by 16.5 mM Nocodazole for 8 hr. The following synchronization protocols were used to measure Plk1 expression in porcine fetal fibroblasts: 1. G0/G1 cells—obtained by reduction of FCS in the culture media to 0.2% for 48 hr; 2. G1/S cells—obtained by stimulation of G0/G1 cells by addition of fresh medium containing 10% FCS and blocking at the beginning of S phase with 6 mM aphidicolin (Sigma Chemical Co.), a DNA polymerase inhibitor, for 14 hr; 3. G2/M cells—produced by washing G1/S cells with PBS (to remove aphidicolin), followed by stimulation with medium containing 10% FCS for 3 hr and finally adding medium containing 10% FCS and 118 mM butyrolactone I (Affinity, Exeter, UK), a cyclin dependent kinase inhibitor, for 5 hr. 4. Partially synchronized cells—obtained by stimulating G0/G1 cells with 10% FCS for 14 or 23 hr, respectively. DNA content was measured by FACS to confirm the cell cycle stage and the efficiency of synchronization as previously described (Kues et al., 2000). Cloning of cDNA of Plks Total RNA was prepared from fibroblasts with the RNeasy Mini kit (Qiagen, Hilden, Germany) and 1 mg of the total RNA was used as template for a reverse transcription (RT) reaction. The RT reaction mix (40 ml total volume) contained: 1 PCR buffer II (Perkin Elmer, Billerica, MA); 5 mM MgCl2 (Gibco-BRL, Gaithersburg, MD); 1 mM dNTPs (USB Corporation, Cleveland, OH); RNase inhibitor 20 U (Perkin Elmer); MuLV RT 2.5 ml (Perkin Elmer); 2.5 mM oligo (dT)12–18 (Gibco-BRL). RT was carried out for 1 hr at 428C with final denaturing for 5 min at 758C. An aliquot of 5 ml of the RT reaction was used as a template for a 100 ml PCR reaction. The primers (Table 1) were based on regions of consensus between the human and mouse Plk’s and modified by adding restriction sites for BamHI and XhoI. The PCR reaction mixture (100 ml total volume) contained: 1 Pfu polymerase reaction buffer (Stratagene, La Jolla, CA); 0.2 mM dNTPs (USB Corporation), cloned Pfu DNA polymerase (Stratagene) 2.5 U; primers 0.5 mM each (MWG Biotech AG, Ebersberg, Germany). PCR temperature cycle: 948C for 45 sec, 608C for 45 sec, 728C for 3 min, repeated for 29 cycles, final extension 728C for 10 min. PCR products were gel purified and sub-cloned into the pGEM1-4Z vector (Promega GmbH, Mannheim, Germany) by using TA cloning strategy pGEM-T Easy kit (Promega GmbH) and sequenced. Semiquantitative RT-PCR Procedures for RNA isolation and the RT reaction were as described above for cloning except that priming of the RT reaction was performed with random hexamers. Relative changes in the abundance of Plk transcripts were determined by a multiplex RT-PCR assay, using either b-actin or GAPDH mRNA as internal standards. Primers used for the quantification are listed in Table 1. The PCR reaction mixture (20 ml) contained: 5 ml of the RT reaction as a template; 1 PCR buffer (Gibco-BRL); 0.2 mM each dNTP (USB Corporation); 1.5 mM MgCl2 (Gibco-BRL); PCR primers 0.5 mM each (MWG Biotech AG); Taq DNA polymerase 0.5 U (GibcoBRL). PCR temperature profile: initial denaturation at 948C for 1 min; then cycles of 948C for 15 sec; 608C for 30 sec; 728C for 1 min; and final elongation 728C for 5 min. Because of the abundance of GAPDH transcript, the PCR mixture initially contained only Plk primers and the GAPDH primers were added after the 5th PCRcycle during a programmed hold at 728C. The total number of PCR-cycles was 27 for Plk1 and 22 for IDENTIFICATION OF POLO-LIKE KINASES IN PORCINE FIBROBLASTS 247 TABLE 1. Sequences of Used Primers Primers A (for cloning) Plk1 sense Plk1 antisense Plk2 sense Plk2 antisense Plk3 sense Plk3 antisense B (for quantification) Plk1 sense Plk1 antisense Plk2 sense Plk2 antisense Plk3 sense Plk3 antisense GAPDH sense GAPDH antisense b-Actin sense b-Actin antisense Product size (bp) GGG ATC CGG CGA AAG AGA TCC CGG AGG TCC TAG T CCT CGA GTT ACG GCT GGC CAG CTC CTT GCA GCA GGG ATC CAT GTG GAA CCC CAA ATT ATC TTT GCT CGA GTC AGC ACC GTC ACT TGA CTG ATG GGG ATC CGC ATC AAG CAG GTT CAT TAC G GCT CGA GTG GGA AGC GAG GTA AGT ACA CTC CTG GAG CTG CAC AAG AGG AGG AA TCT GTC TGA AGC ATC TTC TGG ATG AG CCG GAC AGA CTC TCT TCC AGC TGT T GGT ACC CAA AGC CAT ATT TGT TGG A TGG AGA TGG GTT TGA AGA AGG T TGG GGT TGT AGT GCA CCG TC GTT CCA GTA TGA TTC CAC CCA CGG CAA GTT TGC CAG CCC CAG CAT CAA AGG TAG AAG AGT GTC GAC AAC GGC TCC GGC A GTC AGG TCC CGG CCA GCC A 455 538 274 763 529 Plk: Polo-like kinase. GAPDH. The number of cycles was optimized separately for each gene during preliminary experiments to obtain linear amplification of the template. Primers used separately gave the same amount of product as in the multiplex reaction. The control reactions for each PCR included one tube without the cDNA template and one tube with RNA that had not been exposed to RT. The PCR products were separated on 1.5% agarose gels (Gibco-BRL); stained with ethidium bromide; and recorded using a Photometrics CCD camera. Densitometric evaluation was performed using Image Master 1D Elite V3 (Amersham Pharmacia, Piscataway, New Jersey). mRNA Quantification by the Ribonuclease Protection Assay Linearized plasmid pGEM-4Z containing Plk1 or GAPDH inserts was used as a template for sense and antisense RNA probe synthesis with a MAXIscriptTM T3/T7 Kit (Ambion, Austin, TX). SP6 polymerase was purchased from New England Biolabs (New England Biolabs GmbH, Frankfurt am, Main, Germany). Probes were labeled with [32P]UTP (800 Ci/mmol, 20 mCi/ml) (Amersham Pharmacia Biotech, Freiburg, Germany). The length of protected fragments was 224 bp for GAPDH and 458 bp for Plk1. After in vitro transcription, full-length transcripts were purified from a polyacrylamide gel. For each reaction, 1 mg of total RNA was used for solution hybridization (RPA II kit, Ambion) with the molar access of the probe determined for each probe separately in preliminary experiments. Solution hybridization was carried out at 428C overnight, subsequently RNA/RNA hybrids were treated by RNase T1 and protected fragments were separated on a 5% polyacrylamide gel. The labeled fragments were detected by autoradiography on a Kodak Biomax ML film (Sigma Chemical Co.). Developed films were recorded with a CCD camera and densitometrically analyzed with Image Master 1D Elite V3 (Amersham Pharmacia). Production of Recombinant Plk1, Antibody Generation, and Western Blotting A 344 bp fragment of porcine Plk1 cDNA corresponding to bases 730–1,074 of GeneBank sequence AF339021 was subcloned into the pET30b (Novagen, Madison, WI) bacterial expression vector. The recombinant plasmid was introduced into competent E. coli (BL21[DE3] LysS, Novagen). A single colony was selected and inoculated into LB media containing kanamycin and chloramphenicol (Sigma Chemical Co.). The bacteria cells were cultured at 378C for 3 hr, then IPTG (Sigma Chemical Co.) was added to a final concentration of 0.3 mM. The bacteria were then cultured for 5 hr at 258C and finally, were harvested by centrifugation. The recombinant protein was isolated by HisBindTM and STagTM purification kits (Novagen). For the production of monoclonal antibodies a standard protocol was used (Harlow and Lane, 1988). Briefly, female Balb/c mice were repeatedly immunized with 30 mg purified soluble recombinant protein. Spleen cells were fused with SP 2/0 mouse myeloma cells in 50% PEG (Sigma Chemical Co.) and the resultant hybridomas were selected and screened by ELISA and Western blotting using the porcine Plk1 fusion protein and porcine fetal fibroblast lysate. The supernatant from clone PL-12 was used in this study. Proteins were separated on 10% SDS–PAGE gels and blotted onto PVDF membranes (Amersham) following the protocol of Harlow and Lane (1988). Membranes were blocked with 5% FCS dissolved in 20 mM TrisHCl, pH 7.6, 0.1% Tween-20, 137 mM sodium chloride (TTBS) (all from Sigma Chemical Co.). Plk1 was detected with 1:50 diluted hybridoma cell culture supernatant. Goat anti-mouse antibodies coupled to horseradish peroxidase (HRP) (Sigma Chemical Co.) were used as secondary antibody. HRP activity was visualized by chemiluminescence using the ECL Plus Western blotting system (Amersham Pharmacia Biotech) and detected by exposure to Kodak BioMax Light-1 film. 248 M. ANGER ET AL. Statistical Analysis The t-test was used to evaluate the observed differences between groups. P values lower than 0.05 were considered to be significant. All statistical analysis was performed using KyPlot software. RESULTS Cloning of Porcine Plks 1, 2, and 3 Three partial Plk sequences, representing the porcine homologous cDNAs of Plk1, Plk2, and Plk3, were isolated from fetal fibroblasts by a RT-PCR approach. Using BLAST (Tatusova and Madden, 1999), the amino acid sequence of the cloned 1,579 bp fragment of porcine Plk1 (GeneBank Acc. No. AF339021) was found to be highly conserved to the published Plk1 sequences of other eukaryotes (Fig. 1). The deduced amino acid sequence of this fragment is 96% identical with human Plk1 (AAA36659), 95% with mouse Plk1 (AAA16071), 82% with Xenopus Plx1 (AAC60017), and 51% with Drosophila melanogaster Polo (NP_524179). BLAST also identified two characteristic Plk domains employing a search of the Conserved Domain Database (Altschul et al., 1997). The first one is an N-terminal serine/threonine protein kinase catalytic domain consisting of the 252 amino acids located between positions 8 and 260 of the amino acid sequence essential for kinase activity. The amino acid homology in this domain in comparison with the corresponding serine/threonine protein kinase catalytic domains of human, mouse, Xenopus, and Drosophila Plk1 is 99, 98, 86, and 65%, respectively, suggesting that it is slightly more conserved than the rest of the sequence. The second conserved domain is the C terminal Polo box distinguishing the Polo kinases from other serine/threonine protein kinases. Two Polo box sequences were located. The first occurred between amino acids 372–435 and the second was found between amino acids 470–523 of porcine Plk1. Figure 1 shows the alignment of Plk1 kinases from porcine, human, mouse, Xenopus, and Drosophila species; the serine/threonine kinase domain and the Polo boxes are underlined. The cloned fragment of porcine Plk2 is 952 bp long (GeneBank Acc. No. AF348424) and the deduced amino acid sequence had 316 amino acid residues. Among vertebrates, the porcine Plk2 amino acid sequence had 99% identity with the homologous human sequence (XP_041712), 97% with the mouse sequence (P53351), and 78% with the Xenopus sequence (AAL30175). No homologues were identified in nonvertebrate organisms. The porcine Plk2 fragment contained a portion of the serine/threonine kinase domain and a Polo box identified by a search against the Conserved Domain Database (Altschul et al., 1997). The cloned fragment of Plk3 (GeneBank Acc. No. AF348425) was 1,007 bp long and its deduced amino acid sequence possessed 335 amino acids. The porcine Plk3 fragment had 92, 86, 53% identical amino acids with corresponding human (XP_046526), mouse (AAC52191), and Xenopus (AAL30177) sequences. In the region corresponding to the fragment sequenced in this study, we found some regions of discontinuity with those vertebrate homologues, which may affect its function. For example, the murine sequence of Plk3 contained gaps of 17 amino acids when compared to the porcine and human sequences. Like the other porcine Plks, the porcine Plk3 contained a serine/threonine kinase domain and the Polo box. Cell Cycle Stage Specific Expression of Plk1, Plk2, and Plk3 Initially, we analyzed the expression levels of Plk1, Plk2, and Plk3 in murine and porcine fetal fibroblasts (Figure 2A,B) during the cell cycle. Serum deprived cell cultures were mitotically stimulated by addition of 10% FCS and the relative abundance of Plk transcripts was determined. For each experiment a FACS analysis was performed (Table 2). Plk2 and Plk3 mRNA levels peaked rapidly within 1 hr of serum stimulation, whereas the highest levels of Plk1 mRNA were found after 18–21 hr of stimulation. In porcine cell cultures, Plk1 mRNA showed the highest changes in expression levels throughout the cell cycle. We further analyzed the changes in the expression levels of Plk1 with our optimized synchronization protocols to compare cells synchronized by serum stimulation with cells synchronized by specific inhibitors of cell cycle progression. The mRNA levels of Plk1 were determined by RT-PCR analysis at five points in the cell cycle (Fig. 3A). Samples were obtained by serum deprivation of porcine fetal fibroblasts (0.2% FCS) for 2 days to block the cells in G0 and then releasing the block by the addition of 10% FCS. Cells were harvested at G0/G1 (without serum stimulation) and at 14 and 23 hr following serum stimulation. The remaining two time points were obtained by releasing the fibroblasts from G0 and blocking them at G1/S or G2/ M transition using aphidicolin and butyrolactone I (Kues et al., 2000). The efficiencies of the cell cycle synchronisations were measured by FACS analysis (Table 3). The lowest levels of Plk1 mRNA were observed in G0 fibroblasts (Fig. 3A). After serum stimulation, a significant increase in Plk1 mRNA was evident at 14, and the highest amount was detected at 23 hr (Fig. 3A). Expression levels of Plk1 mRNA in cells reentering the cell cycle following serum stimulation were compared with fibroblasts at G1/S and at G2/M transition using RT-PCR. These results were confirmed by RPA (Fig. 4). The amount of Plk1 mRNA was significantly higher (P 0.01) in cells entering S phase (stimulated with 10% FCS for 14 hr) than in cells blocked at G1/S transition by growth in 10% FCS followed by aphidicolin treatment for 14 hr. Significantly higher Plk1 mRNA was found in cells stimulated with FCS for 23 hr compared with cells blocked at the beginning of S phase by aphidicolin or stimulated for only 14 hr by FCS (P 0.05) Expression of Plk1 Protein in Course of Cell Cycle A monoclonal antibody against porcine Plk1 was generated, to determine, whether the cell cycle dependent IDENTIFICATION OF POLO-LIKE KINASES IN PORCINE FIBROBLASTS 249 Fig. 1. Alignment of Polo-like kinase 1 (Plk1) sequences. The deduced amino acid sequence of porcine Plk1 (AAK28550) aligned with the homologous sequences of man (AAA36659), mouse (AAA16071), Xenopus (AAC60017), and Drosophila (NP_524179). The conserved residues are in black columns. The sequences of the serine/threonine kinase catalytic domain (residues 8–260 of the porcine sequence) and the Polo boxes (residues 372–435 and 470–523 of the porcine sequence) are underlined. transcript levels of Plk1 reflect different protein levels. A recombinant Plk1 fragment (bases 730–1,074), that showed the greatest divergence compared to porcine Plk2 and Plk3 was used to immunize mice. The monoclonal antibody PL-12 was found to be specific for Plk1 and identified a single band of apparently 68 kDa on Western blots from porcine fetal fibroblasts (Fig. 3B). PL-12 antibody did not show reactivity with Plk2 or Plk3 250 M. ANGER ET AL. (data not shown). Western blot analysis using the antibody PL-12 showed that the cell cycle stage dependent translation of the protein (Fig. 3B) followed the same pattern as RT-PCR had revealed for transcription, i.e., Plk1 was first detected at G1/S transition, increased in cells entering S phase (after 14 hr of FCS stimulation), and reached its highest level after G2/M transition when cells were already in mitosis (after 23 hr of FCS stimulation). We also tested the reactivity of the antibody with cell lysates from different species and found that it crossreacts with Plk1 from mouse STO fibroblasts, human HeLa cells and bovine skin fibroblasts (Fig. 3C). Fig. 2. Expression profiles of Plks in murine and porcine fetal fibroblasts. A: Summarized Plk1, Plk2, and Plk3 mRNA expression levels from three independent experiments with mouse fetal fibroblasts. The black bars represent Plk1, gray bars represent Plk2 and white bars represent Plk3. The error bar represents the SD value for each experiment. B: Plk1, Plk2, and Plk3 mRNA expression levels from a single experiment with porcine fetal fibroblasts. Semiquantitative RT-PCR analysis was repeated three times for this experiment with similar results. DISCUSSION Plks appear to be crucially involved in cell cycle regulation in several eukaryotic species (Nigg, 1998). In the present study, we cloned and sequenced cDNA fragments of porcine Plk1, Plk2, and Plk3. While our RTPCR cloning strategy did not allow to clone full-length cDNA, 1,579 bp of sequence were obtained for porcine Plk1; 952 bp for porcine Plk2; and 1,007 bp for porcine Plk3. All three showed a high degree of sequence homology with Plks from other species with the closest similarity to their corresponding human Plks. We identified two domains shared by all three cloned porcine Plks, a catalytic domain typical for serine/threonine kinases, and the Polo box domain, which is the hallmark of all Polo kinases (Clay et al., 1997; Lee et al., 1998). The highest conservation between pig, human, mouse, frog, and fruit fly Plks was found in the catalytic and Polo box domains. Initial experiments on the cell cycle dependent expression of Plk genes in mouse and porcine fetal fibroblasts showed that the regulation of transcription differs dramatically between Plk genes. Plk2 and Plk3 mRNA levels peaked rapidly within 1 hr of serum stimulation of serum deprived cells, whereas the highest Plk1 levels were found after 18–21 hr. In porcine fetal fibroblasts, the greatest changes during the course of TABLE 2. Effects of Serum Deprivation and Serum Stimulation on the Percentages (SD) of Cells in the Different Cell Cycle Stages G0/G1 phase cells S phase cells G2/M phase cells 86.3 6.1 88.2 6.5 76.5 5.4 53.2 7.1 45.4 5.1 44.9 4.7 4.7 2.3 5.3 2.9 12.9 2.0 35.4 1.5 31.2 1.8 21.0 3.6 9.0 3.7 6.5 3.8 10.2 6.0 11.4 2.3 23.4 1.7 34.1 1.6 83.62 85.63 81.69 67.28 38.11 31.60 5.5 4.88 6.82 21.88 50.43 31.59 10.88 9.49 11.49 10.85 11.46 31.60 a A. Mouse fetal fibroblasts 3 days starved 1 hr stimulated 11 hr stimulated 16 hr stimulated 18 hr stimulated 21 hr stimulated B. Porcine fetal fibroblastsb 3 days starved 1 hr stimulated 4 hr stimulated 18 hr stimulated 21 hr stimulated Nocodazole a b Summarized FACS data of three independent experiments. FACS data of a single experiment. IDENTIFICATION OF POLO-LIKE KINASES IN PORCINE FIBROBLASTS 251 Fig. 3. Plk1 expression in synchronized porcine fibroblasts. A: mRNA expression in serum deprived (G0/G1), fetal calf serum (FCS) stimulated (14 and 23 hr) and cells synchronized by aphidicolin (G1/S) or butyrolactone I (G2/M). The gel picture shows representative results of the semiquantitative multiplex RT-PCR. B: Plk1 protein expression in synchronized porcine fetal fibroblasts. The Plk-1 protein was detected by Western blotting with monoclonal antibody PL-12. Cells were treated as was indicated above at RT-PCR analysis. C: Crossreactivity of PL-12 antibody with Plk1 from mouse STO cells, human HeLa cells and bovine skin fibroblasts (BF). cell cycle were found for Plk1. To characterize Plk1 expression in more details, optimized cell cycle synchronization protocols were employed. Under normal culture conditions, 66% of the cells had a DNA content characteristic of G0/G1 and a complete cell cycle lasted approximately 22 hr. Serum starvation increased the proportion of cells in G0/G1 to 80%. The aphidicolin block protocol produced a population in which 78% of cells were blocked at G1/S transition, being unable to initiate DNA synthesis. FCS stimulation for 14 hr after serum starvation resulted in a population of cells in which 49% had DNA content characteristic of G1 and 38% had entered S phase. The protocol with aphidicolin followed by butyrolactone I produced a population in which 41% of the cells were blocked at the G2/M transition and 38% were in G1. FCS stimulation of starved cells for 23 hr produced a population in which 81% of the cells were either in late M phase or early G1. We previously reported that porcine fetal fibroblasts released from blocking with aphidicolin and/or butyrolactone I show no residual effect on proliferation rates or on Plk1 mRNA expression (Kues et al., 2000). The human Plk1 promoter contains domains similar to those found in the promoters of other cell cycle regulated proteins, including cyclin A and cyclin B which are known to be activated at G1/S transition (Uchiumi et al., 1997). This is consistent with the observation that cells blocked by aphidicolin at the G1/S transition had considerably more Plk1 mRNA than the starved cells in G0/G1. Our data for porcine fetal fibroblasts are also consistent with those for cultured mouse and human cells (Lake and Jelinek, 1993; Golsteyn et al., 1994) and with results (Uchiumi et al., 1997) in which various Plk1 promoter—luciferase transgene constructs were employed in HeLa cells synchronized by a double thymidine block. Cells initiated Plk1 transcription at the G1/S transition. These data from human cells coincide with the expression profile of Plk1 in porcine fetal fibroblasts. Interestingly, cells released from the aphidicolin block were able to complete S phase in a significantly shorter time period than starved cells TABLE 3. Cell Cycle Synchronization of Porcine Fetal Fibroblasts (SD) Treatment of cells Unsynchronized 24 hr G0/G1 G1/S 14 hr FCS G2/M 23 hr FCS G0/G1 phase cells a 66.0 6.0 80.3 3.5b 77.7 5.1a,b 49.0 8.7c,d 38.3 2.1d 46.3 4.2c S phase cells a 15.3 3.5 6.0 3.0b 6.0 1.0b 38.0 9.8 20.3 1.2a,c 19.3 3.2a,c G2/M phase cells 18.3 3.1a 13.7 5.5a 16.3 4.2a 13.0 1.7a 41.3 3.2b 34.3 4.9b Summarized FACS data of three independent experiments. The variability between groups was tested and the data within columns, which are significantly different are marked by different superscripts. FCS, fetal calf serum. 252 M. ANGER ET AL. Plk1 protein affords a third alternative for cell cycle analysis. It also requires more material and time than RT-PCR but obviously G0 cells contain no Plk1 protein while early G1 cells contain residual Plk1 protein from the previous cell cycle. Thus protein analysis is the most accurate method for the identification of cells at G0 phase. For a rapid evaluation of synchronization efficiency or the proliferation status of cultured cells, especially when starting material is limited, RT-PCR measurement of Plk1 expression is the method of choice. In conclusion, results of the study reported here, indicate that expression of Plk1 is a suitable marker for determining cell cycle stage in porcine fetal fibroblasts and may thus be a useful tool in the future improvement of porcine somatic nuclear transfer. Fig. 4. Comparative quantification of Plk1 transcripts employing RT-PCR and RPA. Semiquantitative multiplex RT-PCR analysis was repeated three times for each of three experiments and results are summarized in gray bars. The black bars represent the three RPA measurements of RNA obtained from single experiment. The RT-PCR expression levels in all groups differ significantly (P 0.05) from each other with the exception of groups 14 hr FCS and G2/M cells. In RPA analysis, all groups were significantly different (P 0.05), except groups 23 hr FCS and G2/M cells. stimulated by FCS (unpublished observation). This suggests that processes unrelated to DNA replication itself are rate limiting in this phase of the cell cycle and that they are able to proceed in the presence of aphidicolin that inhibits the primer elongation step of DNA replication. Apparently Plk1 expression is a marker for dividing cells. The protein was undetectable and the mRNA was only barely detectable in serum deprived cells arrested in G0/G1. The apparent background of S and G2/M phase cells in serum deprived cultures seems to reflect the fact that early passages of primary cells were used. It is well described that initially primary cultures consist of subpopulations that differ substantially in their potential to divide (Rubin, 1998). The consequence is that even at early passage numbers a certain percentage of cells stops dividing in a stochastic manner and eventually undergoes senescence. However, when the proportion of cells in G0/G1 decrease during mitotic stimulation, the amount of both Plk1 mRNA and protein increased. Highest levels of Plk1 mRNA and protein were found in populations with the highest proportion of cells in mitosis; the two FCS stimulated populations and the population blocked at the G2/M transition. Plk1 expression can be used to characterize the level of synchronization in cells frequently used for a wide spectrum of in vitro techniques, in particular somatic nucleus transfer. In the present experiments, the levels of Plk1 mRNA determined by RT-PCR which is based on enzymatic amplification of the target molecule were compared to data obtained by RPA which is based on hybridization. The results obtained by these two methods were nearly identical. However, RPA requires more starting material and more time than RT-PCR. Immunodetection of ACKNOWLEDGMENTS The authors thank Doris Herrmann and Lenka Travnickova for excellent technical assistance in the laboratory. We are grateful to Dr. Josef Fulka for discussion and critical reading of manuscript. We also thank Dr. Jiri Kanka for bovine skin fibroblasts. REFERENCES Abrieu A, Brassac T, Galas S, Fisher D, Labbe JC, Doree M. 1998. The Polo-like kinase Plx1 is a component of the MPF amplification loop at the G2/M-phase transition of the cell cycle in Xenopus eggs. J Cell Sci 111:1751–1757. 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First, University of Wisconsin, Madison, WI, April 5, 2004 (received for review January 15, 2004) The enzyme telomerase is active in germ cells and early embryonic development and is crucial for the maintenance of telomere length. Whereas the different length of telomeres in germ cells and somatic cells is well documented, information on telomere length regulation during embryogenesis is lacking. In this study, we demonstrate a telomere elongation program at the transition from morula to blastocyst in mice and cattle that establishes a specific telomere length set point during embryogenesis. We show that this process restores telomeres in cloned embryos derived from fibroblasts, regardless of the telomere length of donor nuclei, and that telomere elongation at this stage of embryogenesis is telomerase-dependent because it is abrogated in telomerase-deficient mice. These data demonstrate that early mammalian embryos have a telomerase-dependent genetic program that elongates telomeres to a defined length, possibly required to ensure sufficient telomere reserves for species integrity. would be consistent with the low efficiency of somatic cloning. If telomere restoration in fact occurs during preimplantation development, it remains to be investigated whether this restoration is related to the cloning process itself or represents a general mechanism during embryogenesis to ensure the presence of adequate telomere reserves for species integrity. To test whether telomere length is regulated during embryogenesis, we have analyzed telomere length at different stages of bovine and mouse early development employing embryos derived from somatic nuclear transfer, in vitro production (in vitro maturation, fertilization, and culture), and conventional breeding. Results show that telomere length is determined at morula to blastocyst transition by a telomerase-dependent mechanism. Telomere elongation is restricted to this stage of development and restores telomeres of fibroblast-derived cloned embryos to normal length. T Methods he enzyme telomerase is active in germ cells and during early embryogenesis (1–5) and is crucial for the maintenance of telomere length and germ cell viability in successive generations of a species (6, 7). Telomere shortening limits the regenerative capacity of cells and is correlated with the onset of cancer, aging, and chronic diseases (8–12). The segregation of telomere length from one generation to the next seems to be essential for normal development and well being in mammals. In line with this hypothesis, telomere shortening in telomerase knockout (mTERC⫺/⫺) mice is correlated with a variety of phenotypes including impaired organ regeneration, chromosomal instability, and cancer (9, 13–16). Telomere length segregation is ensured by the active elongation and maintenance program in germ cells. Mature germ cells possess significantly longer telomeres compared to somatic cells (17), possibly because of telomere elongation during germ cell maturation (18). In contrast to telomere elongation in germ cells, little is known about telomere length regulation during embryogenesis. Establishment of telomere length during embryogenesis could determine telomere reserves in newborns. In newborn mice derived from crossbreeding of late generation mTERC⫺/⫺ mice and mTERC⫹/⫺ mice, telomere function is rescued correlating with the elongation of critically short telomeres (19). Similarly, mice derived from intercrosses of mouse strains with different telomere lengths show a telomerase-dependent elongation of those telomeres derived from the strain with shorter telomeres (20, 21). These observations can be explained only if the telomeres are elongated during embryogenesis. In line with this hypothesis, studies on cloned mice and cattle have revealed telomere length restoration of cloned animals even when senescent fibroblasts had been used as the donor cells (22–25). In contrast, cloned sheep derived from epithelial cells had shorter telomeres than control animals (26). Telomere length rescue in cloned animals seems to depend on the used donor cell type (27). Alternatively, rather than telomere restoration during embryogenesis, cloned animals may be derived from the selective propagation of cells with enhanced telomere reserves during the cloning process and兾or early embryonic development, which 8034 – 8038 兩 PNAS 兩 May 25, 2004 兩 vol. 101 兩 no. 21 Quantitative Fluorescence in Situ Hybridization (Q-FISH) on Interphase Nuclei. Q-FISH was performed on interphase nuclei as described on nuclei of adult and fetal fibroblasts (11, 28). Nuclei were isolated by cell lysis with 0.075 M KCl and fixed in glacial methanol兾acetic acid (3:1). Bovine adult and fetal fibroblasts and a human transformed kidney cell line (Phoenix cells, a gift from Gary Nolan, Stanford University, Stanford, CA) were analyzed in parallel with Q-FISH and Southern blotting to validate Q-FISH data. Results correlated closely between the two methods. To ensure linearity of the Q-FISH method, telomere length of liver cells from different generations of telomerase-deficient mice were analyzed by Q-FISH showing the expected decrease in telomere length from one generation to the other (data not shown). Telomere Restriction Fragment (TRF) Length Analysis. TRF were determined in skin biopsies and blood samples (WBC) of cloned and conventionally produced cattle as well as bovine in vitro matured oocytes (see below) and bovine semen as controls. TRF length analysis was done as described in ref. 28. The mean TRF length was calculated by measuring the signal intensity in 10 squares covering the entire TRF smear. All calculations were performed with PCBAS and EXCEL (Microsoft) computer programs. Telomeric Repeat Amplification Protocol. To measure telomerase activity in early embryos, a telomeric repeat amplification protocol assay was performed with a TRAPeze telomerase detection kit (Intergen, Purchase, NY) according to recommendations of the manufacturer. Abbreviations: Q-FISH, quantitative fluorescence in situ hybridization; TRF, telomere restriction fragment. §To whom correspondence should be addressed. E-mail: [email protected] or rudolph. [email protected]. © 2004 by The National Academy of Sciences of the USA www.pnas.org兾cgi兾doi兾10.1073兾pnas.0402400101 In Vitro Production of Bovine Embryos. Bovine embryos were produced as described in refs. 29 and 30. Briefly, viable cumulus– oocyte complexes derived from abattoir ovaries were matured in vitro in groups of 15–20 in 100 l of TCM-199 supplemented with 10 IU of pregnant mare serum gonadotropin and 5 IU of human chorionic gonadotropin (Suigonan, Intervet, Tönisvorst, Germany) and 0.1% BSA (Sigma, A7030) under silicone oil in a humidified atmosphere composed of 5% CO2 in air at 39°C for 24 h. Matured cumulus–oocyte complexes were fertilized in vitro employing 1 ⫻ 106 sperm per ml of frozen兾thawed semen from one bull with proven fertility in in vitro fertilization (29) during a 19-h coincubation under the same temperature and gas conditions as described for in vitro maturation. Presumptive zygotes were cultured up to the morula stage (day 6 after insemination) or blastocyst stage (day 8 after insemination) in synthetic oviduct fluid medium supplemented with BSA, in a mixture of 5% O2, 90% N2, and 5% CO2 (Air Products, Hattingen, Germany) in modular incubator chambers (ICN). Generation of Nuclear Transfer-Derived Embryos. Bovine fetal fibroblasts were obtained from a 61-day female bovine fetus after evisceration and decapitation. Adult fibroblasts were established from ear skin biopsies of an adult female animal collected from a local abattoir. The fibroblasts used for nuclear transfer in these experiments were from passages 2–4 and were induced to enter a period of quiescence (presumptive G0) by serum starvation for 2–3 days (0.5% FCS). Donor cells and cloned animals showed an identical microsatellite pattern (not shown). For enucleation and nuclear transfer, cytochalasin B (7.5 g兾ml) was added to TCM-air. For fusion, 0.285 M mannitol containing 0.1 mM MgSO4 and 0.05% BSA was used. Embryos were reconstructed, and cloned offspring were produced as described in ref. 32.¶ Briefly, in vitro matured oocytes were enucleated by aspirating the first polar body and the metaphase II plate. The donor cells were pelleted and resuspended in TCM-air and remained in this medium until insertion. A single cell was sucked into a 30-m (outer diameter) pipette and was then carefully transferred into the perivitelline space of the recipient oocyte. Cell fusion was induced with 1–2 DC pulses of 0.7 kV兾cm for 30 s each generated by a Kruess electrofusion machine (CFA 400, Hamburg, Germany). At 27 h after onset of maturation, the reconstructed embryos were chemically activated by incubation in 5 M ionomycin (Sigma) in TCM-199 for 5 min followed by a 3–4-h incubation in 2 mM 6-dimethylaminopurine (Sigma) in TCM-199 at 37°C. After three washes, the embryos were cultured for 6 (morulae) or 8 (blastocysts) days as described above. Morulae and blastocysts were used in Q-FISH for determination of telomere length. Production of Mouse Embryos. Wild-type and telomerase knockout (mTERC⫺/⫺, generation 2) females were killed on days 3 (eight-cell and morula stage) or 4 (blastocyst stage) of pregnancy. Day 0.5 is the morning after insemination. The embryos were collected from the oviducts or the uterus as described (33). Statistical Programs. Student’s t test and GRAPHPAD INSTAT software were used to calculate statistical significances and standard deviations. ¶Lucas-Hahn, A., Lemme, E., Hadeler, K. G., Sander, H. G. & Niemann, H. (2002) Theriogenology 57, 433 (abstr.). Schaetzlein et al. Fig. 1. Telomere length analysis of bovine germ cells and primary fibroblasts used for cloning. (A) Distribution of the mean telomere length as determined by Q-FISH in different nuclei of adult fibroblasts (total mean, 10.84 ⫾ 5.73 kb; n ⫽ 118), (B) fetal fibroblasts (total mean, 14.61 ⫾ 4.58 kb; n ⫽ 40), and (C) oocytes (total mean, 16.95 ⫾ 2.51 kb; n ⫽ 13). (D and E) The length of TRFs was determined by Southern blotting on DNA of bovine fetal fibroblasts, adult fibroblasts, and semen. (D) Histogram on the mean value of TRF length as determined in triplicate. Telomere length was significantly shorter in both fibroblast cell lines (adult fibroblasts, 12.03 ⫾ 0.46 kb; fetal fibroblasts, 13.37 ⫾ 0.46 kb) used for cloning compared to bovine semen (15.83 ⫾ 0.41 kb) used for in vivo or in vitro fertilization. (E) Representative radiograph of a Southern blot showing the TRF smear of adult bovine fibroblasts and semen. Dotted line in A–C indicates mean value. All experiments were conducted according to the German animal welfare law and comply with international regulations. Results Telomere Length in Bovine Preimplantation Embryos at the Morula and Blastocyst Stage. We analyzed telomere length regulation during early embryogenesis in bovine embryos and animals derived from cloning or conventional production to elucidate whether the embryo has a defined telomere-elongation program. Q-FISH (Fig. 1 A–C) and Southern blot (Fig. 1E) analysis revealed significantly shorter telomeres in both adult and fetal fibroblasts that had been used for somatic nuclear transfer compared to bovine oocytes and semen serving as controls (Fig. 1 C and E). The telomere length of the adult fibroblasts was shorter than that of fetal fibroblasts (Fig. 1 A, B, and D). Telomere length was analyzed in morula stages, which are known to be telomerase negative or to possess minimum telomerase activity (3–5). Q-FISH analysis revealed significantly shorter mean telomeres in cloned morulae compared to their counterparts derived from in vivo or in vitro fertilization (Fig. 2). The finding of similar telomere lengths in morulae derived from in vivo and in vitro fertilization ruled out the possibility that the in vitro culture per se had a significant effect on telomere length (Fig. 2 A, PNAS 兩 May 25, 2004 兩 vol. 101 兩 no. 21 兩 8035 DEVELOPMENTAL BIOLOGY In Vivo Production of Bovine Embryos. In vivo grown bovine embryos were collected from superovulated donor animals inseminated with semen from the same bull as used for in vitro fertilization. Morulae and blastocysts were nonsurgically recovered from the genital tract of donors employing established protocols at days 6 and 8 after artificial insemination, respectively (31). Fig. 3. Telomere length of cloned and fertilized bovine blastocysts by Q-FISH. (A) Representative photographs of telomere spots in nuclei of blastocysts (day 8 of development) derived from cloning of adult and fetal fibroblasts and in vitro fertilization. Magnification bar, 20 m. (B–D) Distribution of mean telomere length over all analyzed nuclei of blastocysts derived from in vitro fertilization (n ⫽ 129) (B), cloning from fetal bovine fibroblasts (n ⫽ 192) (C), and cloning from adult bovine fibroblasts (n ⫽ 126) (D). (E) Mean value of the mean telomere lengths determined for individual blastocysts derived from in vitro fertilization (19.53 ⫾ 4.6 kb, n ⫽ 6) and from cloning using fetal fibroblasts (17.3 ⫾ 2.66 kb, n ⫽ 6) and adult fibroblasts (21.67 ⫾ 3.92 kb, n ⫽ 5). Note that telomeres were elongated in all groups compared to the morula stage and that in contrast to the differences in telomere length at the morula stage, there was no significant difference between the different groups of blastocysts. (F) A sequential analysis of telomere length in embryos derived from a single in vitro fertilization experiment showed telomere elongation at the morula– blastocyst transition, with an average of 15.8 ⫾ 3.1 kb for morula stages (n ⫽ 6) and 28.7 ⫾ 7.4 kb for blastocyst stages (n ⫽ 6). Dotted line in B–D indicates mean value. Fig. 2. Telomere length of cloned and fertilized bovine morulae by Q-FISH. (A–D). Distribution of mean telomere length over all analyzed nuclei of morulae (day 6 of embryonic development) derived from in vivo fertilization (n ⫽ 234) (A), in vitro fertilization (n ⫽ 118) (B), cloning from adult bovine fibroblasts (n ⫽ 203) (C), and cloning from fetal bovine fibroblasts (n ⫽ 134) (D). (E) Mean value of the mean telomere lengths determined for individual morulae derived by cloning from fetal fibroblasts (8.71 ⫾ 1.81 kb, n ⫽ 8) or adult fibroblasts (9.47 ⫾ 2.07 kb, n ⫽ 6) from in vivo fertilization (12.42 ⫾ 1.37 kb, n ⫽ 7) or in vitro fertilization (14.36 ⫾ 2.67 kb, n ⫽ 4). The difference in telomere length between morulae derived from in vivo and in vitro fertilization was not significant. Dotted line in A–D indicates mean value. B, and E). Telomeres were shorter in all groups of morulae compared to donor cells or germ cells, indicating that there was no significant selection in favor of cells with longer telomere reserves during the cloning process. Next, telomere lengths were analyzed in blastocysts derived from in vitro fertilization or somatic cloning. In all groups, blastocysts had significantly elongated telomeres relative to those at the morula stage (Fig. 3), indicating that telomeres are elongated at this particular stage of bovine embryogenesis. Similarly, a sequential analysis of telomere length in embryos derived from a single in vitro fertilization experiment showed significant telomere elongation in 8036 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0402400101 embryos of an identical genetic background at the morula– blastocyst transition (Fig. 3F). In accordance with previous studies, we did not detect telomerase activity at the morula stage. In contrast, a strong up-regulation of telomerase was observed at the blastocyst stage (3–5), indicating that telomere elongation correlated with the timing of telomerase reactivation during embryogenesis. Telomere lengths of bovine blastocysts were similar regardless of whether they were derived from somatic cloning or in vitro fertilization (Fig. 3). These data provide experimental evidence for an embryo-specific telomere elongation program, which leads to restoration of telomere length in both cloned embryos and embryos produced by in vivo or in vitro fertilization. In line with these observations, telomere length analysis on 1- to 2-year-old cloned cattle derived from fetal or adult donor cells revealed a similar telomere length when measured in WBC or ear biopsies compared to those of age-matched control animals (data not shown). Because WBC are derived from the telomerasepositive hematopoietic compartment, these data indicate that telomere elongation in cloned cattle is restricted to a defined stage of embryogenesis and does not result in abnormally long telomeres in telomerase-positive compartments. The telomere length of WBC and ear biopsies was comparable between clones derived from adult or fetal fibroblasts, indicating that the cell source did not significantly affect telomere length in the cloned offspring. Telomere Elongation During Early Mouse Embryogenesis Is Telomerase-Dependent. To test whether the observed telomere elongation is a general mechanism in mammalian development and whether it Schaetzlein et al. Fig. 4. Telomere length in embryos from mTERC⫺/⫺ and mTERC⫹/⫹ mice by Q-FISH. (A) The distribution of mean telomere fluorescence units (fu) of all analyzed nuclei from wild-type embryos at the 8-cell兾morula stage (1118 ⫾ 484 fu, n ⫽ 274) and at the blastocyst stage (1501 ⫾ 753 fu, n ⫽ 681). (B) Mean value of the mean telomere fluorescence intensity determined for individual embryos at the 8-cell兾morula stage (1,066 ⫾ 406 fu, n ⫽ 32) and at the blastocyst stage (1,350 ⫾ 565 fu, n ⫽ 31). (C) The distribution of mean telomere fu of all nuclei from telomerase knockout embryos (mTERC⫺/⫺) at the 8-cell兾morula stage (1,060 ⫾ 503 fu, n ⫽ 96) and at the blastocyst stage (949 ⫾ 388 fu, n ⫽ 199). (D) Mean value of the telomere fluorescence intensity determined for individual embryos at the 8-cell兾morula stage (995 ⫾ 356 fu, n ⫽ 14) and at the blastocyst stage (877 ⫾ 348 fu, n ⫽ 10). Note that mTERC⫹/⫹ embryos showed telomere elongation at morula兾blastocyst transition, whereas embryos from telomerase-deficient mice (mTERC⫺/⫺) did not show a lengthening of telomeres but rather a slight decrease in telomere length. (E and F) Representative photographs of telomere spots in nuclei of mTERC⫹/⫹ (E) and mTERC⫺/⫺ (F) embryo at 8-cell stage兾morula and blastocyst stage of embryonic development. Magnification bar, 10 m. (G) Mean value of the telomere fluorescence intensity determined for individual embryos at the 8-cell兾morula stage, blastocyst stage, day 8.5兾10.5 (1,329 ⫾ 353 fu, n ⫽ 5), and day 13.5 (1,573 ⫾ 133 fu, n ⫽ 5). Analysis revealed no significant increase in telomere length at these postimplantation stages (P ⬎ 0.05). Dotted line in A and C indicates mean value. Discussion The discovery of telomere elongation at the morula–blastocyst transition in embryos from two mammalian species as well as embryos derived from either in vivo or in vitro fertilization and even somatic nuclear transfer indicates a general program in preimplantation development. Morula–blastocyst transition is a critical step in preimplantation development, leading to first differentiation into two cell lineages, the inner cell mass and the trophoblast. This coincides with a dramatic change in morphology from the compacted morula to the cavity-filled blastocyst stage and is associated with massive changes in embryonic gene expression (32). Our study shows that telomeres of cloned embryos derived from fetal or adult fibroblasts are normalized by the telomere elongation program at the morula–blastocyst transition, indicating that this program resets telomere length to a specific set point. Fibroblasts are a predominantly used donor cell type in somatic nuclear transfer in farm animals. It remains to be determined why telomere length in cloned embryos derived from epithelial cell lines is not always restored (27). Telomeres are not elongated in mTERC⫺/⫺ mouse embryos, indicating that the telomerase enzyme is required for telomere elongation during early embryogenesis. These data coincide with previous reports on haploinsufficiency of the telomerase RNA component (TERC) in mTERC⫹/⫺ mice, leading to offspring from intercrosses of mouse strains with different telomere lengths (20, 21). However, because telomerase activation alone does not necessarily result in telomere elongation in vitro (34), other factors such as telomere binding proteins could be involved in telomere length regulation at this stage of embryogenesis (35). Interestingly, the telomere elongation program discovered in the present study correlates with activation of telomerase at the same stage of human embryogenesis (4). In bovine-cloned and parthenogenetic embryos, telomerase activity followed the same pattern with a significant up-regulation at the blastocyst stage (36). Telomere elongation during embryogenesis could be critical to ensure normal telomere length segregation from one generation to the next and could have direct effects on regeneration, aging, and carcinogenesis during postnatal life. A detailed understanding of this embryonic telomere-elongation program could ultimately be important for the use of cell transplantation and gene therapy approaches for the treatment of degenerative disorders. is telomerase-dependent, we analyzed telomere length in early embryogenesis of mTERC⫺/⫺ and mTERC⫹/⫹ mice. 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Kues and Heiner Niemann Department of Biotechnology, Institut für Tierzucht, Mariensee, D-31535 Neustadt, Germany Farm animals and their products have a longstanding and successful history of providing significant contributions to human nutrition, clothing, facilitation of labour, research, development and medicine and have thus been essential in improving life expectancy and human health. With the advent of transgenic technologies the potential of farm animals for improving human health is growing and many areas remain to be explored. Recent breakthroughs in reproductive technologies, such as somatic cloning and in vitro embryo production, and their merger with molecular genetic tools, will further advance progress in this field. Here, we have summarized the contribution of farm animals to human health, covering the production of antimicrobial peptides, dietary supplements or functional foods, animals used as disease models and the contribution of animals to solving urgent environmental problems and challenges in medicine such as the shortage of human cells, tissues and organs and therapeutic proteins. Some of these areas have already reached the level of preclinical testing or commercial application, others will be further advanced only when the genomes of the animals concerned have been sequenced and annotated. Provided the necessary precautions are being taken, the transmission of pathogens from animals to humans can be avoided to provide adequate security. Overall, the promising perspectives of farm animals and their products warrant further research and development in this field. Farm animals have made significant contributions to human health and well-being throughout mankind’s history. The pioneering work of Edward Jenner with cowpox in the 18th century paved the way for modern vaccination programs against smallpox, as well as other human and animal plagues. To date, more than 250 million people have benefited from drugs and vaccines produced by recombinant technologies in bacteria and various types of mammalian cells and many more will benefit in the future (New Medicines in Development for Biotechnology, 2002; www.phrma.org/newmedicines/biotech/). Further examples of the significant contribution of farm animals to human health are the longstanding use of bovine and porcine insulin for treatment of diabetes as well as horse antisera against snake venoms and antimicrobial peptides. In addition, farm animals are models for novel surgical strategies, testing of biodegradable implants and sources of tissue replacements, such as skin and heart valves. Progress in transgenic technologies has allowed the generation of genetically modified large animals for applications in agriculture and biomedicine, such as the production of recombinant proteins in the mammary gland and the generation of transgenic pigs with expression of human complement regulators in xenotransplantation research [1]. Further promising application perspectives will be developed when somatic cloning with genetically modified donor cells is further improved and the genomes of farm animals are sequenced and annotated. The first transgenic livestock were born less than 20 years ago with the aid of microinjection technology [2]. Recently the first animals with knockout of one or even two alleles of a targeted gene were reported (Table 1). Somatic nuclear transfer has been successful in 10 species, but the overall Table 1. Milestones (live offspring) in transgenesis and reproductive technologies in farm animals Year Milestone Strategy Refs 1985 1986 1997 1997 1998 1998 2000 2002 2002 2003 2003 First transgenic sheep and pigs Embryonic cloning of sheep Cloning of sheep with somatic donor cells Transgenic sheep produced by nuclear transfer Transgenic cattle produced from fetal fibroblasts and nuclear transfer Generation of transgenic cattle by MMLV injection Gene targeting in sheep Trans-chromosomal cattle Heterozygous knockout in pigs Homozygous gene knockout in pigs Transgenic pigs via lentiviral injection Microinjection of DNA into one pronucleus of a zygote Nuclear transfer using embryonic cells as donor cells Nuclear transfer using adult somatic donor cells Random integration of the construct Random integration of the construct Injection of oocytes with helper viruses Gene replacement and nuclear transfer Additional artificial chromosome One allele of a-galactosyl-transferase knocked out Both alleles of a-galactosyl-transferase knocked out Gene transfer into zygotes via lentiviruses [2] [91] [92] [93] [94] [95] [96] [15] [34,35] [36] [97] Corresponding author: Heiner Niemann ([email protected]). www.sciencedirect.com 0167-7799/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2004.04.003 Review TRENDS in Biotechnology Vol.22 No.6 June 2004 287 Table 2. Efficiency of somatic cloning of mammals a Species Number of animalsa % Viable offspring Comments Cattle Sheep Goat Mouse Pig Cat Rabbit Mule Horse Rat , 3000 , 400 , 400 , 300 , 200 1 6 1 1 2 15 –20 5 –8 3 ,2 ,1 ,1 ,1 ,1 ,1 ,1 Up to 30% of the cloned calves showed abnormalities, such as increased birth weight Same problems as with cattle clones Minor health problems reported Some adult mice clones showed obesity and a reduced life span Some cloned piglets had reduced birth weights Estimated total numbers of mammals derived from somatic cloning since 1997. efficiency is low and few cloned offspring have been born worldwide (Table 2). Compared with microinjection of DNA constructs into pronuclei of zygotes, somatic nuclear transfer is superior for the generation of transgenic animals (Table 3). Here, we have summarized the contribution of farm animals to human health covering (i) the production of pharmaceuticals; (ii) production of xenografts for overcoming the severe shortage of human organs and tissues; (iii) the use of farm animals as disease models; (iv) the production of dietary supplements or functional foods; and (v) the contribution of farm animals to solving environmental problems. Farm animals for pharmaceutical production Gene ‘pharming’: production of recombinant human proteins in the mammary gland of transgenic animals The conventional production of rare human therapeutic proteins from blood or tissue extracts is an inefficient, expensive, labour and time consuming process, which in addition bears the risk of contamination with human pathogens. The production of human therapeutic proteins by recombinant bacteria or cell cultures has alleviated these problems and has made several therapeutic proteins available for patients. However, these recombinant systems have several limitations. They are only suitable for ‘simple’ proteins, the amount of protein produced is limited, and post-translational modifications are often incorrect leading to immune reactions against the protein. In addition, the technical prerequisites are challenging and production costs are high. Farm animals such as cattle, sheep, goats, pigs and even rabbits [3,4] have several significant advantages for the production of recombinant proteins over other systems, including their potential for large-scale production, correct glycosylation patterns and post-translational modifications, low running costs, rapid propagation of the transgenic founders and high expression stability. These attractive perspectives led to the development of the ‘gene pharming’ concept, which has been advanced to the level of commercial application [5]. The most promising site for production of recombinant proteins is the mammary gland, but other body fluids including blood, urine and seminal fluid have also been explored [6]. The mammary gland is the preferred production site mainly because of the quantities of protein that can be produced and the ease of extraction or purification of the respective protein. Based on the assumption of average expression levels, daily milk volumes and purification efficiency, 5400 cows would be needed to produce the 100 000 kg of human serum albumin (HSA) that are required per year worldwide, 4500 sheep would be required for the production of 5000 kg a-antitrypsin (a-AT), 100 goats for 100 kg of monoclonal antibodies, 75 goats for the 75 kg of antithrombin III (ATIII) and two pigs to produce 2 kg human clotting factor IX. All these values are calculated on a yearly basis [3]. Large amounts of numerous heterologous recombinant proteins have been produced by targeting expression to the mammary gland via mammary gland-specific promoter elements. Proteins were purified from the milk of transgenic rabbits, pigs, sheep, goats and cattle. The biological activity of the recombinant proteins was assessed and therapeutic effects have been characterized [3,7]. Products such as ATIII, a-AT or tissue plasminogen activator (tPA) are advanced to clinical trials (Table 4) [5]. Phase III trials for ATIII have been completed and the protein is expected to be on the market within the next 2 –3 years. In February 2004 an application was submitted to Table 3. Advantages and disadvantages of two gene transfer methodologiesa Integration efficiency Integration site Gene deletion Construction size Technical feasibility Mosaicism Expression screen in vitro Expression pattern Multi-transgenics a Microinjection Somatic nuclear transfer þ Random 2 . 50 kb (Artificial chromosomes) Technically demanding þþ þ þ Variable þ þþ þ Random or targeted þþ þ , 30 –50 kb Technically demanding 2 þþ þ Controlled, consistent þþ þ Abbreviations: 2, not possible; þ, weak advantage; þþ , moderate advantage; þ þþ , strong advantage www.sciencedirect.com 288 Review TRENDS in Biotechnology Vol.22 No.6 June 2004 Table 4. Proteins produced in the mammary gland of transgenic farm animalsa a Protein Developmental phase Production species Therapeutic application Potential market introduction date AT III TPA a-AT Phase III Phase II/III Phase II/III Goat Goat Goat and/or sheep 2005 . 2006 . 2007 hFVIII HAS Various antibodies Experimental Phase I Phase I/II Sheep Cattle Goat Genetic heparin resistance Dissolving coronary clots Lung emphysema Cystic fibrosis Hemophilia A Blood substitute . 2008 . 2008 . 2007 Abbreviations: a-AT, a-A1-antitrypsin; AT III, antithrombin III; hFVIII, human clotting factor VIII; HSA, human serum albumin; TPA, tissue plasminogen activator the European Market Authorization to allow Atrynw, the recombinant ATIII from the milk of transgenic dairy goats, to enter the market as a fully registered drug. The enzyme a-glucosidase from the milk of transgenic rabbits has Orphan drug registration and has been successfully used for the treatment of Pompe’s disease [8]. This is a rare glycogen storage disorder, which is fatal in children under 2 years and currently application with recombinant a-glucosidase is the only way to treat this metabolic defect. Biologically active human lactoferrin has been produced in large amounts in the mammary glands of transgenic cows and will probably be developed as a biopharmaceutical for prophylaxis and treatment of infectious diseases [9]. Guidelines developed by the Food and Drug Administration (FDA) of the USA require monitoring of the animals’ health, validation of the gene construct, characterization of the isolated recombinant protein, as well as performance of the transgenic animals over several generations. This has been taken into account when developing ‘gene pharming’, for example by using only animals from prion disease-free countries (New Zealand) and keeping the animals in very hygienic conditions. Successful drug registration of Atrynw will demonstrate the usefulness and solidity of this approach and will accelerate registration of further products from this process, as well as stimulate research and commercial activity in this area. When considering the ‘gene pharming’ concept, one has to bear in mind that not every protein can be expressed at the desired levels. Erythropoietin (EPO) could not be expressed in the mammary gland of transgenic cattle [10] and was even detrimental to the health of rabbits transgenic for EPO [11]. We have shown that human clotting factor VIII (hFVIII) cDNA constructs can be expressed in the mammary gland of transgenic mice, rabbits and sheep [1,12]. However, the yields of biologically active recombinant hFVIII protein from ovine milk were low because hFVIII was rapidly sequestered into ovine milk. [13]. These results show that the technology needs further improvements to achieve high-level expression with large genes having complex regulation, such as that coding for hFVIII, although higher levels of hFVIII have been reported in transgenic swine [14]. With the advent of transgenic crops that produce pharmacologically active proteins, there is an array of recombinant technologies available that will allow the most appropriate production system for a specific protein to be targeted. An interesting new development is the generation of transchromosomal animals (Table 1). A human artificial www.sciencedirect.com chromosome (HAC) containing the entire sequences of the human immunoglobulin heavy and light chain loci has been introduced into bovine fibroblasts, which were then used in nuclear transfer. Transchromosomal offspring were obtained that expressed human immunoglobulin in their blood. This system could be a significant step forward in the production of human therapeutic polyclonal antibodies [15]. Further studies will show whether the additional chromosome will be maintained over future generations and how stable expression will be. Production of a new class of antibiotics: cationic antimicrobial peptides With increasing antibiotic resistance in bacterial species, there is a growing need to develop new classes of antimicrobial agents. Cationic anti-microbial peptides (AMP) have many of the desired features [16] because they possess a broad spectrum of activity, kill gram-positive and gram-negative bacteria rapidly, are unaffected by classical resistance genes and are active in animal models [17 – 19]. AMPs belong to the innate immune defense, which acts as a first barrier ahead of humoral and cellular immune systems, and neutralizes bacteria by interacting specifically with their cell membranes. Their low transmembrane potential of , -100 mV, and the abundant anionic phospholipids are essential for this selective interaction. It is proposed that AMPs physically disintegrate the cell membrane, and in addition can interact with several intracellular target molecules [16]. More than 500 such peptides have been discovered in plants, insects, invertebrates, fish, amphibians, birds and mammals [16 – 20]. AMPs from livestock species would be superior antimicrobial drugs because they would lack cytotoxic effects that were found for insect peptides; the evolution of resistance would not affect the human specific innate immunity [20]. Prominent examples of cationic anti-microbial peptides from farm animals already in advanced clinical trials (Phases II– III) are Iseganan (derived from protegrin-1 peptide of pig leucocytes; Intrabiotics; http://intrabiotics. com/) and MBI-594 (similar to indolicidin peptide from bovine neutrophils; Micrologix, http://mbiotech.com/). To date, there are no documented cases of antimicrobial peptide-resistance for AMPs and combinatorial approaches in peptides (20 possible amino acids in each position) provide great potential for rational drug design [21]. Recombinant production will keep the production costs low. Review TRENDS in Biotechnology Xenotransplantation of porcine organs to human patients Solid organs Today . 250 000 people are alive only because of the successful transplantation of an appropriate human organ (allotransplantation). On average, 75 – 90% of patients survive the first year after transplantation and the average survival of a patient with a transplanted heart, liver or kidney is 10– 15 years. This progress in organ transplantation technology has led to an acute shortage of appropriate organs, and cadaveric or live organ donation cannot cover the demand in western societies. The 2001 figures from the United Network for Organ Sharing (www. unos.org) in the USA show that the ratio of patients with organ transplantation to those on the waiting list is , 1:4 (Table 5). A similar ratio is found in other countries such as the UK, France and Germany. A new person is added to the waiting list every 14 minutes. This has led to the sad and ethically challenging situation in which several thousand patients who could have survived if appropriate organs had been available die every year. To close the growing gap between demand and availability of appropriate organs, porcine xenografts are considered the solution of choice [22,23]. Today the domesticated pig is considered the optimal donor animal because (i) the organs are similar in size to human organs; (ii) porcine anatomy and physiology are not too different from that of humans; (iii) pigs have short reproduction cycles and large litters; (iv) pigs grow rapidly; (v) maintenance of high hygienic standards is possible at relatively low costs; and (vi) transgenic techniques for modifying the immunogenicity of porcine cells and organs are well established. The process of generating and evaluating transgenic pigs as potential donors for xenotransplants involves a variety of complex steps and is time-, labour- and resourceintensive. Essential prerequisites for successful xenotransplantation are: (i) Overcoming the immunological hurdles. (ii) Prevention of transmission of pathogens from the donor animal to the human recipient. (iii) Compatibility of the donor organs with the human organ in terms of anatomy and physiology. The immunological obstacles in a porcine-to-human xenotransplantation are the hyperacute rejection response (HAR), acute vascular rejection (AVR), cellular rejection and potentially chronic rejection [24]. The HAR Table 5. Overview of transplanted organs and demand for organ transplantation (USA)a a Type of transplant Transplantations in 2001 Waiting patients Kidney Liver Pancreas Kidney/Pancreas Heart Heart/Lung Lung Total 14 152 5177 468 884 2202 27 1054 23 964 52 772 17 520 1318 2520 4163 209 3799 79 845 Data from United Network for Organ Sharing, 2001 (www.unos.org). www.sciencedirect.com Vol.22 No.6 June 2004 289 occurs within seconds or minutes. In the case of a discordant organ (e.g. in transplanting from pig to human) naturally occurring antibodies react with antigenic structures on the surface of the porcine organ and induce HAR by activating the complement cascade via the antigen– antibody complex. Ultimately, this results in the formation of the membrane attack complex (MAC). However, the complement cascade can be shut down at various points by expression of regulatory genes that prevent the formation of the MAC. Regulators of the complement cascade are CD55 (decay accelerating factor, DAF), CD46 (membrane cofactor protein, MCP) or CD59. MAC disrupts the endothelial cell layer of the blood vessels, which leads to lysis, thrombosis, loss of vascular integrity and ultimately to rejection of the transplanted organ [24]. Induced xenoreactive antibodies are thought to be responsible for AVR, which occurs within days of a transplantation of a xenograft; disseminated intravascular coagulation (DIC) is a predominant feature of AVR. Despite severe immunosuppressive treatment, a disturbed thrombocyte function and DIC were observed in a pig-toprimate xenotransplant model [25,26]. The endothelial cells of the graft’s microvasculature loose their antithrombic properties, attract leucocytes, monocytes and platelets leading to anemia and organ failure. The underlying mechanism for DIC and thrombotic microangiopathy is thought to be activation of the endothelial cells attributed to incompatibilities between human and porcine coagulation factors [25]. At least three incompatibilities between human and porcine coagulation systems have been identified; the first is the failure of porcine thrombomodulin (TM) to activate human anticoagulant protein C, the second is that the porcine tissue factor pathway inhibitor fails to inhibit human clotting factor Xa and the third is that porcine von Willebrand factor (vWF) binds and activates human platelets [26]. Human thrombomodulin (hTM) and heme-oxygenase 1 (hHO-1) are crucially involved in the etiology of DIC and might be good targets for future transgenic studies to improve long-term survival of porcine xenografts by creating multi-transgenic pigs. The cellular rejection occurs within weeks after transplantation. In this process the blood vessels of the transplanted organ are damaged by T-cells, which invade the intercellular spaces and destroy the organ. This rejection is observed after allotransplantation and is normally suppressed by life-long administration of immunosuppressive drugs [24]. When using a discordant donor species such as the pig, overcoming the HAR and AVR are the preeminent goals. The most promising strategy for overcoming the HAR is the synthesis of human complement regulatory proteins (RCAs) in transgenic pigs [22,23,27,28]. Following transplantation, the porcine organ would produce the complement regulatory protein and can thus prevent the complement attack of the recipient. Pigs transgenic for DAF or MCP have been generated by microinjection of DNA constructs into pronuclei of zygotes. Hearts and kidneys from these animals have been transplanted either heterotopically, (in addition to the recipient’s own organ) or 290 Review TRENDS in Biotechnology Vol.22 No.6 June 2004 Table 6. Success rates of RCA-transgenic porcine organs after transplantation to primate recipientsa a RCA Organ/kind of transplant Recipient Immuno-suppression Survival (days) hDAF hDAF hDAF hDAF hCD59 hCD46 Heart/heterotropic Heterotopic Orthopic Kidney/orthotopic Kidney/orthotopic, heterotopic Heart, heterotopic Cynomologus Cynomologus Baboon Cynomologus Cynomologus Baboon þþþ þþ þþþ þþ þþþ þþ , 135 , 90 , 28 , 90 , 20 , 23 Abbreviations: þ , weak immunosuppression; þþ , moderate immunosuppression; þþ þ, heavy immunosuppression; RCA, regulator of complement activity orthotopically (life supportive) into non-human primates. Survival rates reached 23 – 135 days with porcine xenografts transgenic for one of the two complement regulators; survival rates were heavily affected by the strength of the immune-suppressive protocol (Table 6) [29 – 31]. Similarly, transgenic expression of hCD59 was compatible with an extended survival of porcine hearts in a perfusion model or following transfer into primates [32,33]. These data show that HAR can be overcome in a clinically acceptable manner by expressing human complement regulators in transgenic pigs [22]. Another promising strategy towards successful xenotransplantation is the knockout of the antigenic structures on the surface of the porcine organ that cause HAR. These structures are known as 1,3-a-gal-epitopes and are primarily produced by activity of 1,3-a-galactosyltransferase (a-gal). Piglets in which one allele of a-gal locus had been knocked out by homologous recombination in primary donor cells that were employed in nuclear transfer were recently generated [34,35]. The birth of four healthy piglets with disruption of both allelic loci for a-gal has meanwhile also been published. Applying toxin A from Clostridium difficile to cells that already carried one deleted a-gal allele selected a cell clone, which carried an inactivating point mutation on the second allele. This cell clone was then used in nuclear transfer [36]. The usefulness of these animals for xenotransplantation has recently been reported [37]. Further improvements in the success of xenotransplantation will arise from the possibility of inducing a permanent tolerance across xenogenic barriers [38,39]. A particularly promising strategy for long-term graft acceptance is the induction of a permanent chimerism via intraportal injection of embryonic stem cells [40]. Prevention of transmission of zoonoses from the donor animal to the human recipient is crucial for clinical application of porcine xenografts. This aspect gained particular significance when a few years ago it was shown that porcine endogenous retroviruses (PERV) can be produced by porcine cell lines and can even infect human cell lines in vitro [41]. However, until today no infection has been found in patients that had received various forms of living porcine tissues (e.g. islet cells, insulin, skin, extracorporal liver) for up to 12 years [42]. Recent intensive research has shown that porcine endogenous retroviruses probably do not present a risk for recipients of xenotransplants provided all necessary precautions are taken [43– 46]. In addition, a strain of miniature pigs has been identified that does not produce infective PERV [47]. Although xenotransplantation poses numerous further challenges to research, it is expected www.sciencedirect.com that transgenic pigs will be available as organ donors within the next five to ten years. Guidelines for the clinical application of porcine xenotransplants are already available in the USA and are currently being developed in several other countries. The ethical challenges of xenotransplantation have been a matter of a worldwide intensive debate. A general consensus has been reached that the technology is ethically acceptable provided the individual well-being does not compromise public health by producing and transmitting new pathogens. Economically xenotransplantation might be viable if the enormous costs caused by patients suffering from severe kidney disease, needing dialysis or those suffering from chronic heart diseases could be avoided by a functional kidney or heart xenograft. Xenogenic cells and tissue Another promising area of application for transgenic animals will be the supply of xenogenic cells and tissue. Several intractable diseases, disorders and injuries are associated with irreversible cell death and/or aberrant cellular function. Despite numerous attempts, primary human cells cannot yet be expanded well enough in culture. In the future, human embryonic stem cells could be a source for specific differentiated cell types that can be used in cell therapy [48,49]. Xenogenic cells, in particular from the pig, hold great promise for successful cell therapies for human patients because cells can be implanted at the optimal therapeutic location (i.e. immunoprivileged sites, such as the brain), genetically or otherwise modified before transplantation to enhance cell function, banked and cryopreserved, or combined with different cell types in the same graft [50]. Xenogenic cell therapy has been advanced to preclinical studies. Porcine islet cells have been transplanted to diabetic patients and were shown to be at least partially functional over a limited period of time [51]. Porcine fetal neural cells were transplanted into the brain of patients suffering from Parkinson’s and Huntington’s disease [52,53]. In a single autopsied patient the graft survived for .7 months and the transplanted cells formed dopaminergic neurons and glial cells. Pig neurons extended axons from the graft site into the host brain [52]. Further examples for the potential use of porcine neural cells are in cases of stroke and focal epilepsy [54]. Olfactory ensheathing cells (OECs) or Schwann cells derived from hCD59 transgenic pigs promoted axonal regeneration in rat spinal cord lesion [55]. Thus, cells from genetically modified pigs could restore electrophysiologically functional axons across the site of a spinal cord transsection. Xenogenic porcine cells could also be useful Review TRENDS in Biotechnology as a novel therapy for liver diseases. On transplantation of porcine hepatocytes to Watanabe heritable hyperlipidemic (WHHL) rabbits (a model for familial hypercholesterolemia) the xenogenic cells migrated out of the vessels and integrated into the hepatic parenchyma. The integrated porcine hepatocytes provided functional low density lipoprotein (LDL) receptors and thus reduced cholesterol levels by 30 – 60% for at least 100 days [56]. A clone of bovine adrenocortical cells restored adrenal function upon transplantation to adrenalectomized severe combined immunodeficient (SCID) mice indicating that functional endocrine tissue can be derived from a single somatic cell [57]. Bovine neuronal cells were collected from transgenic fetuses, and when transplanted into the brain of rats resulted in significant improvements in symptoms of Parkinson’s disease [58]. Furthermore, xenotransplantation of retinal pigment epithelial cells holds promise for treating retinal diseases such as macular degeneration, which is associated with photoreceptor losses. Porcine or bovine fetal cardiomyocytes or myoblasts might provide a therapeutic approach for the treatment of ischemic heart disease. Similarly, xenogenic porcine cells might be valuable for the repair of skin or cartilage damage [50]. In light of the emergence of significantly improved protocols for genetic modification of donor animals and new powerful immunosuppressive drugs xenogenic cell therapy will evolve as an important therapeutic option for the treatment of human diseases. Farm animals as models for human diseases In mouse genetics the generation of knockout animals is a standard procedure and several thousand strains carrying gene knockouts or transgenes have been developed [Mouse Knockout and Mutation Database (MKMD); http:// research.bmn.com/mkmd]. Models have been developed for several human diseases. However, mouse physiology, anatomy and life span differ significantly from those in humans, making the rodent model inappropriate for several human diseases. Farm animals, such as pigs, sheep or even cattle could be more appropriate models to study human diseases in particular non-insulin-dependent diabetes, cancer and neurodegenerative disorders, which require longer observation periods than those possible in mice [59 – 61]. With the aid of the microinjection technology an important pig model for the rare human eye disease Retinitis pigmentosa (PR) has been developed [62]. Patients with PR develop night blindness early in life attributed to a loss of photoreceptors. The transgenic pigs express a mutated rhodopsin gene and show a great similarity with the human phenotype. Treatment models with value for human patients are being developed [63]. The development of the somatic cloning technology and the merger with targeted genetic modifications and conditional gene expression will enhance the possibilities for creating useful models for human diseases in large animals. A good example is the knockout of the prion gene that would make sheep and cattle non-susceptible to spongiform encephalopathies (scrapie and BSE). Mice models showed that the knockout of the prion protein is the only secure way to prevent infection and transmission of the disease [64]. The first successful targeting of the ovine www.sciencedirect.com Vol.22 No.6 June 2004 291 prion locus has been reported; however the cloned lambs carrying the kockout locus died several days after birth [65]. Prion knockout animals could be an appropriate model for studying the epidemiology of spongiform encephalopathies in humans and are crucial for developing strategies to eliminate prion carriers from a farm animal population. The pig could be a useful model to study defects of growth hormone releasing hormone (GHRH), which is characterized by a variety of conditions such as Turner syndrome, hypochrondroplasia, Crohn’s disease, intrauterine growth retardation or renal insufficiency. Application of recombinant GHRH in an injectable form and its myogenic expression has been shown to alleviate these problems in a porcine model [66]. An important aspect of large animal models for human diseases is the recent finding that somatic cloning per se does not result in shortening of the telomeres. Telomeres are highly repetitive DNA sequences at the end of the chromosomes that are crucial for their structural integrity and function and are thought to be related to lifespan. Telomere shortening is usually correlated with severe limitations of the regenerative capacity of cells, the onset of cancer, ageing and chronic disease with significant impact on human lifespan [67 – 70]. Expression of the enzyme telomerase, which is primarily responsible for the formation and rebuilding of telomeres, is suppressed in most somatic tissues postnatally. Although telomeres in cloned sheep (Dolly) derived from epithelial cells were shorter than those of naturally bred age matched control animals, telomere lengths in cloned mice and cattle were not different from those determined in age matched controls even when senescent donor cells had been employed [71 – 75]. Recent studies in our laboratory have revealed that telomere length is established already early in preimplantation development by a specific genetic programme and is dependent on telomerase activity [76]. Dietary modifications of animal products Application of gene and biotechnology for nutrition and biomedicine is more developed for plants than farm animals [77]. The term nutriceuticals in the farm animal context means that gene and biotechnology are used to enhance farm animal products to improve diet and have concomitant medical applications. Because these products have a proven pharmacological effect on the body, they are regarded as ‘drugs’ and must be tested for efficacy and safety. Functional foods are those designed to provide specific and beneficial physiological effects on human health and welfare and should prevent diet-related diseases. Functional foods from animals could be used to lower cholesterol levels in an effort to battle cardiovascular diseases, to reduce high blood pressure by adding angiotensin converting enzyme (ACE) inhibitors or to increase immunity by adding specific immunostimulatory peptides [78]. However, data showing that nutriceuticals are really beneficial for human health are rare. Regarding the production of improved quality animal products, interesting observations have been made in several beef cattle breeds, such as Belgian Blue and Piedmontese. These breeds were accidentally bred for 292 Review TRENDS in Biotechnology Vol.22 No.6 June 2004 mutations of the myostatin gene, which renders it nonfunctional or less functional than the wild-type gene [79]. The mutated genes cause muscle hypertrophy and led to improved meat quality. This observation makes targeted modifications of the myostatin gene an interesting option for the meat industry. A diet rich in non-saturated fatty acids is correlated with a reduced risk of stroke and coronary diseases. One transgenic approach to this is the generation of pigs with increased amounts of nonsaturated fatty acids. Pigs producing a higher ratio of unsaturated versus saturated fatty acids in their muscles are currently under development in Japan. An attractive example for targeted genetic modification could be dairy production [80,81]. Apart from conventional dairy products, it could be possible to produce fat-reduced or even fat-free milk or milk with a modified lipid composition via modulation of enzymes involved in lipid metabolism; to increase curd and cheese production by enhancing expression of the casein gene family in the mammary gland; to create ‘hypoallergenic’ milk by knockout of the b-lactoglobulin gene; to generate lactose-free milk via knockout of the a-lactalbumin locus that is the key molecule in milk sugar synthesis; to produce ‘infant milk’ in which human lactoferrin is abundantly available or to produce milk with a highly improved hygienic standard via an increased level of lysozyme or other anti-microbiological substances in the udder. Lactosereduced or lactose-free milk could make dairy products suitable for consumption by a large proportion of the world’s population who do not possess an active lactase enzyme in their gut system. However, one has to bear in mind that lactose is the main osmotically active substance in milk and a lack thereof could interfere with milk secretion. A lactase construct has been expressed in the mammary gland of transgenic mice and reduced lactose contents by 50 – 85% without altering milk secretion [82]. However, mice with a homozygous knockout for alactalbumin could not feed their offspring because of the high viscosity of the milk [83]. These diverging findings demonstrate the feasibility of obtaining significant alterations of milk composition by applying the appropriate strategy. The physicochemical properties of milk are mainly affected by the ratio of casein variants. Therefore, casein is a prime target for the improvement of milk composition. Mouse models have been developed for most of the above modifications indicating the feasibility of obtaining significant alterations in milk composition but at the same time showing that unwanted side effects cannot be ruled out [83,84]. Only one full-scale study in livestock has been reported as yet [85]. The recent report showed that the casein ratio can be altered by overexpression of b- and k-casein in cattle clearly underpinning the potential for improvements in the functional properties of bovine milk [85]. environmental pollution. These pigs carry a bacterial phytase gene under the transcriptional control of a salivary gland specific promoter, which allows the pigs to digest plant phytate. Without the bacterial enzyme, the phytate phosphorus passes undigested into manure and pollutes the environment. With the bacterial enzyme, the fecal phosphorus output was reduced up to 75% [86]. These environmentally friendly pigs are expected to enter the commercial production chains within the next few years. Towards environmentally friendly farm animals Phosphorus pollution by animal production is a serious problem in agriculture and excess phosphate from manure promotes eutrophication. Phytase transgenic pigs have been developed to address the problem of manure-based References www.sciencedirect.com Precautions and perspectives Throughout mankind’s history farm animals have made significant contributions to human health and well-being. The convergence of the recent advances in reproductive technologies with the tools of molecular biology opens a new dimension for this area [1]. Major prerequisites will be the continuous refinement of reproductive biotechnologies and a rapid completion of livestock genome sequencing and annotation. The technology developed in the deciphering of the human genome will improve and accelerate sequencing of genomes from livestock [87]. We anticipate genetically modified animals will play a significant role in the biomedical arena, in particular via the production of valuable pharmaceutical proteins and the derivation of xenografts, within the next 5 – 7 years. Agricultural application might be further away (. 10 years) given the complexity of some of the economically important traits and the public skepticism of genetic modification related to food production [1]. A crucial aspect of animal-derived products is the prevention of transmission of pathogens from animals to humans. This requires sensitive and reliable diagnostic and screening methods for the various types of pathogenic organisms. The recent findings (see above) that the risk of PERV transmission is negligible are promising and show that with targeted and intensive research such important questions can be answered within a limited period of time, paving the way for preclinical testing of xenografts. Furthermore, it should be kept in mind that the biomedical applications of farm animals will require strict standards of ‘genetic security’ and reliable and sensitive methods for the molecular characterization of the products. A major contribution towards the goal of well-defined products will come from array technology (cDNA, peptide or protein arrays), which establishes ‘fingerprint’ profiles at the transcriptional and/or protein level [88,89]. Meanwhile improvements of RNA isolation and unbiased amplification of tiny amounts of mRNA (picogram) enable researchers to analyse RNA from single embryos [90]. With the aid of this technology one can gain in-depth insight into the proper functioning of a transgenic organism and thereby ensure the absence of unwanted side effects [88,89]. This would also be required to maintain the highest possible levels of animal welfare in cases of genetic modification. 1 Niemann, H. and Kues, W.A. 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DOI 10.1095/biolreprod.104.031229 Isolation of Murine and Porcine Fetal Stem Cells from Somatic Tissue1 Wilfried A. Kues, Björn Petersen, Wiebke Mysegades, Joseph W. Carnwath, and Heiner Niemann2 Department of Biotechnology, Institut für Tierzucht, Mariensee, D-31535 Neustadt, Germany ABSTRACT Adult stem cells have been previously isolated from a variety of somatic tissues, including bone marrow and the central nervous system; however, contribution of these cells to the germ line has not been shown. Here we demonstrate that fetal somatic explants contain a subpopulation of somatic stem cells (FSSCs), which can be induced to display features of lineageuncommitted stem cells. After injection into blastocysts, these cells give rise to a variety of cell types in the resultant chimeric fetuses, including those of the mesodermal lineage; they even migrate into the genital ridge. In vitro, FSSCs exhibit characteristics of embryonic stem cells, including extended self-renewal; expression of stem cell marker genes, such as Pou5f1 (Oct4), Stat3, and Akp2 (Tnap) and growth as multicellular aggregates. We report that fetal tissue contains somatic stem cells with greater potency than previously thought, which might form a new source of stem cells useful in somatic nuclear transfer and cell therapy. assisted reproductive technology, early development, embryo, environment, fibroblasts, germ line, Oct4, pluripotency, stem cells INTRODUCTION Adult stem cells with a surprisingly high degree of plasticity have been identified among hematopoietic and mesenchymal cell populations from bone marrow and the ependymal and subventricular zones of the central nervous system, as well as in vitro-derived cultures thereof [1–8]. These cells are capable of contributing to organs of the three germ layers, but contribution to the germ line has not yet been shown. Recently, it has become a matter of debate whether these cells have inherent pluripotency or whether fusion with differentiated cells can account for the observed plasticity [9–12]. Fibroblasts are the principal cell type in connective (mesodermal) tissue, where they mediate structural and functional processes, such as formation of a collagen meshwork and wound healing [13–15]. Primary fibroblasts can be obtained from subdermal and other connective tissues [16, 17]. These cells represent a poorly characterized mixture of cell lineages [18], in which the composition varies with the developmental stage of the donor and the tissue of origin [14]. The regenerative ability of connective tissue suggests the presence of tissue-specific progenitor cells. In the present study, we have tested whether primary fibroblast cultures contain stem cells with the potential for multilineage differentiation. To identify rare stem cells Supported by DFG. Correspondence: FAX: 0 5034 871 101; e-mail: [email protected] 1 2 Received: 26 April 2004. First decision: 11 May 2004. Accepted: 9 December 2004. Q 2005 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org within a large population of primary somatic cells, we used the OG2 transgenic mouse line, which carries the Oct4 (also known as Pou5f1) promoter driving a green fluorescent protein (GFP) marker [19, 20]. Oct4 is a transcription factor of the POU family that is crucial for maintenance of embryonic stem cells in the undifferentiated state. It plays a major role in mouse embryogenesis [21–23]. Oct4 regulates expression of several genes, including Fgf4, Rex1 (also known as Zpf42), Sox2, Ssp1 (osteopontin), Hand1 and Ifnt1 [23, 24]. The Oct4 promoter is active in oocytes, zygotes, early cleavage-stage embryos, the inner cell mass of the blastocyst and in embryonic carcinoma and stem cells [21, 24, 25]. Oct4 is downregulated when cells leave the germ line and is found exclusively in germ cells in fetal and adult mice [21–23]. (Re)activation of the Oct4 gene in somatic cells would be a strong indication that these cells fulfill an essential molecular precondition for pluripotency. To test the potential for multilineage differentiation and functional contribution to organogenesis, the most informative assay is injection of the putative stem cells into recipient blastocysts and the generation of chimeras. Here, we report the identification of a fetal somatic stem cell (FSSC) population in primary fibroblast cultures derived from two mammalian species (mouse and pig) and show that simple changes of the culture conditions allow selective enrichment of the FSSCs. The FSSCs could be more amenable to reprogramming and would thus be a new source of donor cells in somatic nuclear transfer or celltherapy approaches. MATERIALS AND METHODS Cell Culture of Fetal and Adult Somatic Tissues Primary cultures were prepared by somatic explant (,1 mm3) cultures of connective tissue. For isolation of fetal cells, porcine fetuses of Postcoitus (PC) Day 25 were eviscerated, the extremities and the heads were removed. Cell cultures from adult pigs were isolated from subdermal tissues of ear clips. The tissues were isolated under sterile conditions and washed twice in phosphate buffered saline (PBS) containing antibiotics and were then cut into small pieces and pasted to cell culture dishes by employing recalcified microdrops of bovine plasma. Bovine plasma was isolated from EDTA-blood of healthy cows by centrifugation (4000 3 g, 15 min) and stored frozen in aliquots at 2808C. For use, an aliquot was thawed, supplemented with 80 mM CaCl2 and microdrops of 5–10 ml were pipetted into a cell culture dish. Small pieces of tissue were placed into the plasma drops, and culture dishes were incubated for 10–30 min at 378C until plasma coagulation was completed. Subsequently, tissues were maintained in Dulbecco modified Eagles medium (DMEM) medium supplemented with 2 mM glutamine, 1% nonessential amino acids, 1% vitamin solution, 0.1 mM mercaptoethanol, 100 U/ml penicillin, 100 mg/ml streptomycin (Sigma, Deisenhofen, Germany) containing 10% fetal calf serum (FCS; batch numbers 40G321K, 40G2810K; Gibco, Karlsruhe, Germany) and incubated in humidified 95% air with 5% CO2 at 378C [16, 17]. Outgrowing cells were trypsinized and subpassaged once before cryopreservation. Typically, first outgrowing cells from fetal explants became visible within 24 h of incubation, and subpassaging was done after 7 days of culture. For high serum culture, the serum content of the standard medium was increased to 30% FCS. For suspension culture, colonies were selectively isolated and completely dissociated in a trypsin solution, then 1020 FETAL SOMATIC STEM CELLS 104 cells were seeded into 35-mm bacteriological dishes. Every second day, 50% of the medium was replaced with new medium. To determine the maximal replicative limit, cultures were serially subpassaged and 12.5 3 103 cells were seeded per square centimeter in six-well dishes, trypsinized after 5–7 days, counted, and reseeded. The number of accumulated population doublings per passage was determined using the equation, PD 5 log(A/B)/log 2, in which A is the number of collected cells and B is the number of plated cells. Murine fibroblasts were obtained from Day 11.5– 15.5 fetuses or adult OG2 mice [14] (homozygous for the Oct4-GFP transgene) or from double transgenic fetuses of crosses of OG2 with Rosa26 mice. Confocal microscopy was applied to detect GFP using a Zeiss Axiomat LSM and an excitation wavelength of 488 nm. Embryonic stem (ES) cells (wildtype GS1 129/Sv) were cultured as described [26]. Animal handling was in accordance with institutional guidelines. In some experiments, porcine FSSCs were cultured in standard medium (DMEM 10% FCS) supplemented with growth factors. As controls, high serum and standard cultures were run in parallel and, after 7 days, the three-dimensional-colony formation and alkaline phosphatase (AP) induction were determined. The following growth factors were supplemented: human leukemia inhibitory factor (LIF, 1000 U/ml; Chemicon International), epidermal growth factor (EGF, 0.1 ng/ml; TEBU GmbH, Offenbach, Germany), platelet-derived growth factor (PDGF, 1.0 ng/ml; TEBU GmbH), basic fibroblast growth factor (bFGF, 1.0 ng/ml; TEBU GmbH), endothelial cell growth factor (ECGF, 500 ng/ml; Roche Diagnostics, Mannheim, Germany), and insulin (250 ng/ml; Sigma). In some experiments, porcine FSSCs were cultured in high serum medium supplemented with one of the following inhibitory peptides: 1) bFGF inhibitory peptide H-1948 (5 mM; Bachem, Weil am Rhein, Germany), 2) insulin-like growth factor (IGF-I) analog H-1356 (10 mM; Bachem), 3) PDGF antagonist H-8650 (10 mM; Bachem), and 4) insulin receptor (689702) H-4212 (10 mM; Bachem). In parallel, high serum and standard cultures were run as controls and, after 7 days, three-dimensional-colony formation and AP induction were determined. DNA contents were measured by fluorescent activated cell sorting as described [16]. In addition, nuclei from FSSCs were karyotyped and the ploidy status was assessed. RT-PCR of Specific mRNAs In brief, total RNA was isolated from cells grown in six-well dishes and 0.5 mg RNA was reverse transcribed (RT) into cDNA using random hexamers in a total volume of 40 ml. Aliqouts of 2.5 ml of the RT-reaction were used as templates for polymerase chain reaction (PCR). Murine Oct4 and Stat3 were amplified by PCR with the following primers and conditions: 59-GGC GTT CTC TTT GGA AAG GTG TTC and 59-CTC GAA CCA CAT CC TTC TCT (35 cycles, annealing temperature 578C) for the murine Oct4; 59-TCA AGC ACC TGA CCC TTA GG and 59-CTG AAG CGC AGT AGG AAG GT (37 cycles, 588C annealing temperature) for Stat3 (U06922). For the amplification of TNAP (AF285233), the following primer pair was used: 59-ATG AGG GTA AGG CCA AGC AG and 59CCA CCA TGG AGA CAT TTT CC (34 cycles, 588C annealing temperature). Porcine OCT4 was amplified with intron-spanning 59-AGG TGT TCA GCC AAA CGA CC and 59-TGA TCG TTT GCC CTT CTG GC primers (AJ251914) and 36 cycles. Glyceraldehyde phosphate dehydrogenase (GAPD) was amplified with the primer pair 59-CCT CCA CTA CAT GGT CTA CAT GTT CCA GTA and 59-GCC TGC TTC ACC ACC TTC TTG ATG TCA TCA (25 cycles, annealing temperature 608C). The GAPD primers matched completely with the porcine ortholog (U48832) and showed a single nucleotide mismatch with the mouse ortholog (BC083065). The PCR reactions were performed in 20-ml volumes, consisting of 20 mM TrisHCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 mM deoxynucleoside triphosphates, 1 mM of specific primer pairs and 0.5 U of Taq DNA polymerase (Gibco). Reactions without RT or without template were used as controls. Measurement of Cell Proliferation by BrdU Incorporation DNA synthesis was measured by 5-bromo-29deoxy-uridine (BrdU) incorporation as described [17]. Incorporated BrdU was detected by a chromogenic immunoassay employing an anti-BrdU antibody conjugated with alkaline phosphatase. Immunohistology For antibody staining, cells were cultured on gelatinized coverslips. Sterile coverslips were placed in six-well cell culture dishes and covered with a 1% gelatine solution (in PBS) for 5 min. The gelatine solution was 1021 then removed and the semidry coverslips were seeded with cells. After 3– 4 days of culture, the cells were fixed in cold 80% methanol. The following monoclonal antibody dilutions were used: anti-vimentin (AMF-17b, 1: 200; Developmental Studies Hybridoma Bank, IA) and anticytokeratin (peptide17, 1:100; Sigma). A rhodamine-labeled secondary anti-mouse antibody (1:2000; Molecular Probes, Netherlands) was used. In some cases, the nuclei were counterstained with 1 mM Hoechst 33342 [27]. The samples were examined with an Olympus BX60 microscope equipped with phase-contrast and epifluorescence optics, using band-pass rhodamine and Hoechst filter sets. Staining of Endogenous Alkaline Phosphatase Activity Standard and high-density cell cultures were prepared in six-well cell cultures dishes. The cultures were washed with PBS, fixed in 3.7% paraformaldehyde for 15 min, washed in PBS, and then incubated in a solution containing 25 mM TrisHC (pH 9.0), 4 mM MgCl2, 0.4 mg sodium-anaphtylphosphate, 1 mg/ml Fast Red TR (Sigma), and 0.05% Triton X100 for 60 min. Chimera Generation by FSSC Injection into Host Blastocysts Rosa26 homozygous mice were obtained from Jackson Laboratory (Bar Harbor, ME) and mated with homozygous OG2 animals to generate double-transgenic fetuses carrying both marker genes, which were used to isolate FSSCs. Day 11.5—15.5 fetuses were isolated and employed for fetal cell cultures using the explant method described above. For blastocyst injections, 6- to 10-wk-old female CD2F1 mice were superovulated with 10 U eCG at noon on Day 22, followed by 10 U hCG on Day 0, and were then mated with CD2F1 males. The next day, females were checked for plug formation. At Day 3.5, females were sacrificed and the uterine tracts were isolated and flushed with PBS containing 1% bovine serum albumin. Blastocysts were isolated and incubated in PBS/1% albumin at 378C. Single blastocysts were transferred into a micromanipulation unit (Zeiss) and fixed with a holding pipette. On average, 2–15 double transgenic cells (OG2/Rosa26) were injected into the blastocoel with the aid of a microcapillary. In total, 8–10 blastocysts were transferred into the uteri of Day 2.5 or Day 3.5 pseudopregnant NMRI females that had been mated with vasectomized males. Fetuses were recovered at Day 10.5–15.5 and either stained for lacZ-positive cells [28] as whole mounts, or dissected and screened for GFP expression in genital ridges and other organs. RESULTS Activation of the Germline-Specific Oct4 Promoter in Somatic Cells Fetal OG2-transgenic mice, which express the GFP gene under transcriptional control of the germ-line-specific Oct4 promoter, were employed for explant cultures. Explants of somatic tissue with an average size of ,1 mm3 were isolated from fetuses at PC Days 11.5, 13.5, and 14.5; attached with microdrops of bovine plasma to cell culture dishes, and cultured in DMEM. Special care was taken to isolate explants from mesodermal tissue of the neck and shoulder regions. As expected, no GFP-positive cells were found in the tissue explants and in the first outgrowths after 2 days in culture (Fig. 1, C and D). However, after 8 days of culture, GFP-positive cells were detected within the outgrowing primary cells (Fig. 1, E and F), indicating (re)activation of the germ-line-specific Oct4-GFP marker cassette. As positive control, the genital ridges were isolated in parallel and primordial germ cells could be unequivocally detected by their strong expression of the Oct4-GFP marker (Fig. 1, A and B). Additional experiments suggested that the microenvironment of the plasma microdrops played an important role in stimulating the proliferation of GFP-positive cells (Fig. 1, G–I). Therefore, subpassages of the outgrowing cells were cultured in DMEM containing high fetal calf serum supplementation (30% FCS) and a population of ;1023 GFP-positive cells (Fig. 1, G–I) could be main- 1022 KUES ET AL. FIG. 1. Activation of Oct4-promoter in somatic explants of Oct4-eGFP tg mice. Genital ridge of a male fetus (PC Day 14.5) with tissue-specific expression of GFP in the primordial germ cells is shown under fluorescent (A) and bright-field optics (B). Bar 5 150 mm. The primordial germ cells show expression of GFP for 8 days in culture (A, B), no outgrowing GFPpositive cells could be detected. Outgrowing cells of a somatic explant, under fluorescent (C) and bright-field (D) optics after 2 days of culture. No GFP-positive cells were found. In the upper left, the explant is visible. After 8 days in culture, several GFP-positive cells were detectable within the outgrowing cells (E, F). Bar 5 140 mm. Confocal analysis of murine FSSCs cultured in medium with high serum supplementation: (G) fluorescent, (H) bright-field, and (I) merged images; bar 5 10 mm. The GFP is preferentially located in the cytoplasm, probably because it doesn’t contain a nuclear localization motif. J) RT-PCR detection of endogenous Oct4, Stat3, and Tnap transcripts in murine FSSCs; M, DNA ladder; lane 1: FSSCs; lane 2: no RT; lane 3: ES cells; lane 4: no RT; lane 5: no template control. tained. Expression of the endogenous Oct4 gene was confirmed by RT-PCR (Fig. 1J). Oct4 is one of the best characterized genes for stemness. The presence of Oct4 transcripts has been shown by two independent assays in this study. The absence of a PCR product from fibroblast cultures argues against the amplification of an Oct4 pseudogene. In addition, the tissue nonspecific variant of alkaline phosphatase (TNAP) and Stat3 were expressed in high serum-supplemented cultures as determined by RT-PCR (Fig. 1); in contrast, no expression of the embryonic or intestinal AP genes could be detected (not shown). In Vivo Differentiation Potential by Injection of FSSCs into Blastocysts To determine the developmental potential, FSSCs were injected into murine blastocysts, which were subsequently transferred to pseudopregnant recipients. FSSCs of both sexes were isolated from double transgenic fetuses of OG2 and Rosa26 mouse strains. These cells carried the germlinespecific Oct4 GFP and the ubiquitously active lacZ reporter gene constructs and thus allowed distinguishing them from the cells of the recipient blastocysts. Day 10.5–15.5 fetuses derived from the injected blas- tocysts were isolated and analyzed for chimerism either by staining for lacZ activity or by fluorescence microscopy to identify GFP-positive cells. Of a total of 19 analyzed fetuses, 7 contained progeny cells from the injected FSSCs (Table 1). Chimerism was detected in mesenchymal organs, such as liver, muscle, and tongue, but also in the genital ridges. Figure 2A shows an example of a chimeric fetus with massive lacZ staining in liver, tongue, and genital ridges, suggesting that at least parts of these organs were derived from the injected cells. Chimeric and wildtype fetuses were derived from embryo transfers that had been performed on the same day, were stained for LacZ activity in parallel, and photographed on the same slide. It is unclear whether the apparent oversize of the chimeric fetus is related to the cell injection. The summarized data for the blastocyst transfer suggest that development of embryos after FSSC injection is compromised (Table 1). Figure 2B shows the presence of GFP-positive cells in the genital ridges of a male PC Day 15.5 fetus. In total, 16 GFP-positive cells were counted in the squeeze preparation, and these cells behaved like prospermatogonia in that they floated within the ducts of the genital ridges. GFP-positive cells were not found in other organs, such as heart, liver, brain, or connective tissue. 1023 FETAL SOMATIC STEM CELLS TABLE 1. Generation of chimeric fetuses by injection of FSSCs into recipient blastocysts.* Transgenic background No. of blastocysts transferred Recovered fetuses 6–8 10–15 2–5 6–8 Rosa26 Rosa26 Rosa26/OG2 Rosa26/OG2 57 6 24 49 4 1 5 9 10–15 Control blastocysts w/o cell injection Rosa26/OG2 wt 20 29 0 14 No. injected FSSCs Chimeric fetuses 0 1 3 2 1 of 4 of 1 of 5 of 7 of 2 — 0 of 9 0 of 5 Assay LacZ: LacZ: LacZ: LacZ: OG2: LacZ: OG2: Positive cells found in none liver, genital ridge, tongue mesoderm, sev. organs, low chimerism mesoderm, sev. organs, low chimerism genital ridge NA some background in spinal cord — * NA, not applicable. Isolation of FSSCs from Porcine Tissues The isolation of FSSCs with stem-like properties from murine somatic explants raised the question whether this is a species-specific phenomenon or whether similar cells could be obtained from other species. Therefore, somatic explants of porcine fetuses (PC Day 25) were established and subpassaged once using standard protocols. Immunostaining showed uniform labeling for vimentin and no labeling for cytokeratin (data not shown), suggesting that most of the cells were fibroblasts. However, culture in DMEM with high serum supplementation activated the porcine OCT4 gene (Fig. 3M). Porcine OCT4 was amplified with intron spanning porcine-specific primers; the obtained PCR product was purified, sequenced, and found to be identical to the corresponding OCT4 cDNA sequence. Control cultures maintained in DMEM/10% FCS did not express detectable OCT4 mRNA levels With high serum concentrations, the cultures grew faster and lost contact inhibition, resulting in the formation of three-dimensional colonies (Fig. 3, A and B). Only cells within the three-dimensional colonies continued to proliferate as measured by BrdU incorporation, whereas cells in the surrounding monolayers were mitotically inactive (Fig. 3D). Staining for endogenous AP activity revealed a massive induction of AP-positive cells, almost exclusively associated with the three-dimensional colonies (Fig. 3, G–J). FIG. 2. Generation of chimeric fetuses by FSSC injection into blastocysts. A) Wholemount staining for LacZ activity in a control fetus (left) and a fetus (PC Day 15.5) derived from a FSSC (Rosa26/OG2)-injected blastocyst (right). Note the b-galactosidase staining in liver (arrow) and genital ridge (arrowheads) of the chimeric fetus. B) Oct4 promoter driven expression of GFP in the genital ridges of a chimeric fetus (PC Day 15.5) derived from a FSSC (Rosa26/OG2)-injected blastocyst (left). Genital ridge from a control OG2/Rosa26 fetus (right). Bar 5 20 mm. C) Higher magnification of a GFP-positive cell (top) in the genital ridge of a chimeric fetus (PC Day 15.5) and corresponding phase-contrast image (bottom). Bar 5 20 mm. 1024 KUES ET AL. FIG. 3. Induction of three-dimensional growth and AP-positive cells in porcine FSSCs. A) Three-dimensional-colony growth of porcine FSSCs (passage 3, 5 days) in culture medium with high serum supplementation, (B) same view as in A with Hoechst 33324-stained nuclei under ultraviolet excitation. Bar 5 20 mm for A– F; it is displayed in D. C) Control culture of the same cell batch cultured in standard medium (10% FCS, 5 days). D) BrdU incorporation in high serum cultures (5 days, 30% FCS). Note that only cells within the three-dimensional colonies (arrows) incorporated BrdU, the surrounding monolayer is unlabelled; inset: another three-dimensional colony. E) BrdU incorporation in proliferating fibroblasts (three-dimensional, standard medium with 10% FCS); the majority of the cells are labeled. F) BrdU incorporation in confluent fibroblasts (5 days, standard medium), the majority of the cells became contact inhibited and stopped proliferating. G–J) Induction of AP-positive cells, accompanied with thredimensional-colony growth after 2, 4, 6, 8 days in high serum culture. K) Higher magnification of AP-positive cells aggregated in three-dimensional colony (4 days). L) Individual AP-positive cells within the fibroblast monolayer. Bars 5 20 mm. M) Species-specific RT-PCR detection of OCT4 transcripts in porcine FSSCs (top); 1, DNA ladder with prominent 600-base pair fragment; lane 2: no template; lane 3: murine ES cells; lane 4: porcine fibroblasts; lane 5: porcine FSSCs. As control, transcripts of the housekeeping gene GAPD were amplified by RT-PCR (bottom). AP-positive cells differed (Fig. 3, K and L) from typical fibroblasts in that they displayed dendritic projections. These changes were not found in control cultures from identical batches of cells when grown under standard conditions with 10% FCS supplementation (Fig. 3C, Table 2). In total, 6.7% of microwells seeded with 10 cells from high serum cultures resulted in continuously growing cultures, suggesting that 1 out of 150 cells was able to initiate clonal growth. Induction of colony growth and AP expression by high serum supplementation were abolished by pretreatment with heat (908C) or trypsin (data not shown). Adult porcine fibroblasts derived from subdermal tissue explants of three animals did not display three-dimensionalcolony growth (Fig. 4) when cultured for 7 days in DMEM/ 30% FCS, but the frequency of AP-expressing cells increased 2- to 10-fold over this period (Table 2). In comparison, when fetal cells were used, a 100- to 1000-fold increase in AP-positive cells was observed. Induction of three-dimensional-colony growth and AP expression in porcine cultures was at least one order of magnitude higher than that seen in murine cultures (Table 2). When high serum cultures were split and one part of the population was returned to standard medium, colony growth ceased and AP-positive cells disappeared in the 1025 FETAL SOMATIC STEM CELLS TABLE 2. High serum induction of AP-positive cells and three-dimensional (3D)-colonies in porcine and murine cell isolates.* High serum induced Age of donors porcine Day 25–27 PC Day 25 PC 0.5–1.5 y murine Day 11.5 PC Day 13.5 PC Day 14.5 PC 4 mo Tissue source AP-positive cells (fold increase compared with standard cultures) 3D colonies (no./6-well) Method n Mesoderm Mesoderm Ear biopsy Try Expl Expl 2 12 3 250–850 100–1000 2–10 100–290 65–340 Mesoderm Mesoderm Mesoderm Subdermal tissue Expl Expl Expl Expl 3 1 3 1 8 3 11 1.5 3–5 0 3–5 0 * Try, Trypsinisation of pooled fetuses (n 5 6–8); Expl, explant cultures from individual fetuses or adult subdermal tissues. standard culture within 2–3 subpassages, suggesting that the induction and proliferation of FSSCs are dependent on high levels of not-yet-identified serum factors (Fig. 4B). When high serum cultures were subpassaged five times in standard medium, the colony growth phenotype was lost and a switch to high serum medium was no longer associated with the appearance of three-dimensional growth, suggesting that the FSSC population had been lost due to unfavorable conditions. To narrow down the possibilities of potential factors in serum responsible for these effects, it was tested whether purified growth factors could substitute for high serum supplementation. Porcine FSSC cultures in standard medium (DMEM 10% FCS) were supplemented with either hLIF, PDGF, FGF1, ECGF, or insulin. However, none of these growth factors induced three-dimensional growth or induction of AP expression compared with approximately 200– 300 colonies in the positive controls (not shown). In addition, no inhibitory effect of the inhibitory peptides FGF1 inhibitory peptide, IGF1 analog, PDGF antagonist, or insulin-receptor [609–702] [29–32] on the FSSC phenotype could be detected; the peptide supplemented cultures developed the same number of FSSC colonies as the positive controls. These data indicate that the examined growth factors and receptors are not involved in the apparent pluripotent phenotype induced by the high serum supplementation. FSSCs Have Increased Proliferative Potential Culture medium supplemented with high serum resulted in a dramatically altered growth curve (Fig. 4C). Porcine FSSC cultures maintained under high serum conditions grew continuously over a period of .120 days and exceeded more than 100 cell doublings without reaching a plateau phase (Fig. 4C). In contrast, standard cultures, i.e., DMEM with 10% FCS, were compatible with only 50–60 cell doublings before mitotic activity ceased after approximately 70 days. The FSSCs maintained a diploid status, as measured by fluorescence-activated cell sorting (Fig. 4D) and metaphase spreads of cells from passage 20. Thirtytwo karyotyped nuclei of porcine FSSC cultures contained the euploid number of 38 chromosomes; only 1 nucleus with 37 chromosomes was found. FSSCs Are Capable of Forming Spheroids and Exhibit Anchorage-Independent Growth To investigate the growth potential of the colony-forming cells, porcine three-dimensional colonies of 200–300 mm diameter were isolated and trypsinized to obtain singlecell suspensions. Subsequently, 104 cells were seeded into bacteriological culture dishes. In DMEM/30% FCS, irregular aggregates consisting of only few cells (2–20), were detected. These cells did not divide and the majority apparently underwent cell death (Fig. 4E). Culture medium supplemented with retinoic acid induced the formation of tiny aggregates, which, after 2–4 days, attached to the surface (Fig. 4F). Supplementation of the culture medium (DMEM/30% FCS) with dexamethasone resulted in aggregation of small multicellular spheroids within 24 h, which continued to grow up to a diameter of .400 mm after 10– 15 days and contained nearly exclusively AP-positive cells (Fig. 4, G and H). Dexamethasone is an antiinflammatory glucocorticoid, which regulates T cell survival, growth, and differentiation and inhibits the induction of nitric oxide synthase. Several protocols for differentiation of stem cells also employed dexamethasone. The dexamethasone-derived spheroids were capable of forming secondary spheroids if trypsinized and reseeded in bacteriological dishes. If plated on gelatinized coverslips, dexamethasone spheroids reattached and monolayer cells grew out. These outgrowing cells were vimentin negative, whereas control cultures kept in standard medium with 10% FCS were strongly positive (Fig. 4, I and J). DISCUSSION The present findings provide compelling evidence for the presence of a somatic stem cell population in explant cultures derived from mouse and pig fetuses. The FSSC cells are characterized by extended proliferative capacity, altered morphology, expression of stem cell-specific markers, including Oct4, Stat3, and Tnap and by contact- and anchorage-independent growth. Chimeric fetuses, obtained by injection of murine FSSCs into recipient blastocysts, showed that the FSSCs were able to contribute to various mesenchymal organs and in particular the genital ridges. Whether FSSCs show a similar broad spectrum of differentiation potential as multipotent adult progenitor cells [6] remains to be shown. As some, albeit few, cells expressed GFP fluorescence driven by the Oct4 promoter in the chimeric genital ridges, germ-line transmission might be possible. The final proof for germ-line transmission by FSSCs would be the generation of live and fertile chimeric animals. The finding that GFP-positive cells were not found outside of the genital ridges indicates that the Oct4 marker was correctly activated in cells committed to the germ line. It also suggests that at least some of the FSSC descendants were 1026 KUES ET AL. FIG. 4. Growth characteristics of FSSCs in vitro. A) Porcine cultures from fetal and adult origin of the same batches, respectively, were split and cultured with high serum (30%) or standard (10% FCS) conditions in six-well plates; after 5 days, the cultures were fixed and stained for endogenous AP activity. Note the massive induction of AP-positive three-dimensional colonies in the fetal culture (red dots). B) The induction of three-dimensional-colony growth and AP expression is serum dependent. After six passages with constant three-dimensional-colony formation and AP expression in high serum (30% FCS), fetal cells were trypsinized, replated, and cultured for two passages with standard medium (10% FCS) before AP staining. C) Growth curves of fetal somatic cells cultured in standard medium (□) containing 10% FCS and high serum medium (l) containing 30% FCS. D) Cell cycle status in standard and high serum culture. Note that the high serum culture displays a normal ploidy. E, F) Anchorage-independent growth of FSSCs in suspension culture. High serum-induced three-dimensional colonies were isolated, trypsinized to single cell suspensions, and seeded into bacteriological dishes to prevent attachment. E) In DMEM with high serum supplementation, tiny aggregates formed. F) In high serum medium with retinoic acid (1027 M), the initial aggregates reattach and show outgrowing cells on the surface. G) In high serum medium with dexamethasone (1027M), spheroids of .300-mm size grow over 10–15 days; inset: lower magnification. H) Dexamethasone-spheroids stained for endogenous AP, bar 5 230 mm. I) Expression of vimentin in fibroblasts cultured in standard medium (passage 5), merged image of antibody (red) and nuclei (blue) staining (left). J) Loss of vimentin epitopes in cells derived from dexamethasone spheroids. After 15 days of suspension culture, the spheroids were allowed to reattach to gelatinized coverslips and probed with a monoclonal antivimentin antibody. capable of migrating into the genital ridge. The relatively low percentage of chimerism might be due to the fact that the cells used for blastocyst injection were not preselected for Oct4-GFP expression. Chimerism was found preferentially in liver, muscle, and tongue. The LacZ-positive cells in the tongue may actually represent lymphatic tissue. It is well known that the fetal liver contains hepatic as well as migratory hematopoietic progenitor cells. However, we have not yet determined which cell types in these three organs are derived from the injected cells. This is the subject of future studies. No chimerism was detected in heart and brain, two organs that showed a high rate of spontaneous cell fusions in a recent study [12]. However, we cannot fully exclude the possibility that fusion with differentiated cells might have contributed to the observed chimerism. FSSCs represent a rare subpopulation in fetal fibroblast cultures, and the proliferation of FSSCs critically depends on the culture conditions, including an autologous feedercell layer and factors present in bovine serum. The explant culture is based on microdrops of bovine plasma as an adhesive support, which seems to be essential for the initial stimulation of FSSC proliferation. Standard culture media compositions, with no or low amounts of FCS supplementation, are associated with a progressive loss of the FSSC phenotype. These observations show that appearance of FSSCs is critically dependent on cell-collection method and/or culture conditions. At present, it is not clear whether FSSCs represent a preexisting subpopulation in the fetus or whether they are the result of a de novo formation induced by the culture conditions. Potential sources are ectopic primordial germ cells [19] or fetal tissue-specific stem cells. In humans, it has been shown that mesenchymal stem cells are present in a variety of tissues during development, whereas in adults, they are predominantly found in the bone marrow [33]. FETAL SOMATIC STEM CELLS Activation of the Oct4 promoter in murine explant outgrowths, although at lower expression levels than in murine ES cells, after a few days in culture suggests that the culture conditions induce this phenotype. We cannot completely rule out that this reprogramming is attributed to dedifferentiation of a susceptible, non-lineage-committed cell population present in fetal tissues. The observed frequency of ;1/1000 GFP-positive cells in the in vitro cultures may actually underestimate the occurrence of FSSCs, as GFP expression was low and might be around the detection limit. Clonal growth was found for 1 out of 150 cells and chimerism was detected in 7 out of 19 fetuses. In line with the expression of stem cell marker genes, FSSCs show three-dimensional-colony growth in adherent culture and spheroid growth in suspension culture, whereas fibroblasts cease dividing and arrest in the G1 phase of the cell cycle under these conditions [34–36]. Loss of contactinhibition and anchorage-independent growth is characteristic for ES cells or transformed cells [37]. Our data provide convincing evidence that, unlike many cell lines derived from tumors or cells transformed by oncogenic agents, the FSSC subpopulation does not result from spontaneous immortalization or transformation. FSSCs do not exhibit a crisis followed by clonal outgrowth, and they show no chromosomal abnormalities or aneuplodies. The altered growth characteristics of FSSCs are reversed after exposure to standard cell culture conditions. Unlike rodent cell cultures, cells from humans and livestock rarely, if ever, immortalize spontaneously [38]. Our findings show that FSSCs can be efficiently and reliably isolated from porcine, but also from bovine and ovine, fetal explants (data not shown) and with a lower efficiency from murine fetal explants. The diminished efficiency of FSSC stimulation from murine explant cultures may be attributed to the species specificity of factors present in bovine serum. In conclusion, this study shows that explants from fetal somatic tissues contain a subpopulation of potentially pluripotent stem cells, which fulfill most of the essential criteria for stem cells, including self-renewal ability, multilineage differentiation, and differentiation in vivo [39]. Therefore, the FSSCs might hold great promise for regenerative therapies. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. ACKNOWLEDGMENTS 23. The authors thank Erika Lemme for advice on blastocyst injection. We gratefully acknowledge critical discussion and the gift of the OG2-transgenic mouse line from Hans Schöler. 24. REFERENCES 25. 1. Weissman IL. Stem cells: units of development, units of regeneration and units of evolution. Cell 2000; 100:157–168. 2. Shih CC, Weng Y, Mamelak A, LeBon T, Hu MC, Forman SJ. Identification of a candidate human neurohematopoietic stem-cell population. Blood 2001; 98:2412–2422. 3. Tropepe V, Coles BL, Chiasson BJ, Horsford DJ, Elia AJ, McInnes RR, van der Kooy D. Retinal stem cells in the adult mammalian eye. Science 2000; 287:2032–2036. 4. Bjornson C, Rietze RL, Reynolds BA, Magli MC, Vescovi AL. Turning brain into blood: a hematopoietic fate adopted by adult neural stem cells in vivo. Science 1999; 283:354–357. 5. 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Delayed embryonic lethality in mice lacking protein phosphatase 2A catalytic subunit Calpha. Proc Natl Acad Sci U S A 1998; 95:12370–12375. Kues WA, Brenner HR, Sakmann B, Witzemann V. Local neurotrophic repression of gene transcripts encoding fetal AChRs at rat neuromuscular synapses. J Cell Biol 1995; 130:949–957. Friedrich G, Soriano P. Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev 1991; 5:1513–1523. Yayon A, Aviezer D, Safran M, Gross JL, Heldman Y, Cabilly S, Givol D, Katchalski-Katzir E. Isolation of peptides that inhibit binding of basic fibroblast growth factor to its receptor from a random phageepitope library. Proc Natl Acad Sci U S A 1993; 90:10643–10647. Pietrzkowski Z, Wernicke D, Porcu P, Jameson BA, Baserga R. Inhibition of cellular proliferation by peptide analogues of insulin-like growth factor 1. Cancer Res 1992; 52:6447–6451. 1028 KUES ET AL. 31. Engstrom U, Engstrom A, Ernlund A, Westermark B, Heldin CH. Identification of a peptide antagonist for platelet-derived growth factor. J Biol Chem 1992; 267:16581–16587. 32. Naranda T, Goldstein A, Olsson L. A peptide derived from an extracellular domain selectively inhibits receptor internalization: target sequences on insulin and insulin-like growth factor 1 receptors. Proc Natl Acad Sci U S A 1997; 94:11692–11698. 33. Caplan AI, Bruder SP. Mesenchymal stem cells: building blocks for molecular medicine in the 21st century. Trends Mol Med 2001; 7: 259–264. 34. Holley RW, Kiernan JA. ‘‘Contact inhibition’’ of cell division in 3T3 cells. Proc Natl Acad Sci U S A 1968; 60:300–304. 35. Todaro GJ, Lazar GK, Green H. The initiation of cell division in a contact-inhibited mammalian cell line. J Cell Physiol 1965; 66:325– 333. 36. Otsuka H, Moskowitz M. Arrest of 3T3 cells in G1 phase in suspension culture. J Cell Physiol 1975; 87:213–220. 37. Layer PG, Robitzki A, Rothermel A, Willbold E. Of layers and spheres: the reaggregate approach in tissue engineering. Trends Neurosci 2002; 25:131–134. 38. Rubin H. Multistage carcinogenesis in cell culture. Dev Biol (Basel) 2001; 106:61–67. 39. Verfaillie C. Adult stem cells: assessing the case for pluripotency. Trend Cell Biol 2002; 12:502–508. MOLECULAR REPRODUCTION AND DEVELOPMENT Bovine ICM Derived Cells Express the Oct4 Ortholog PREM S. YADAV,1,2 WILFRIED A. KUES,1 DORIS HERRMANN,1 JOSEPH W. CARNWATH,1 1 AND HEINER NIEMANN * 1 Department of Biotechnology, Institute for Animal Breeding (FAL) Mariensee, Neustadt, Germany 2 Department of Physiology and Reproduction, Central Institute for Research on Buffaloes, Hisar, Haryana, India ABSTRACT The goal of this study was to define conditions for the successful isolation of embryonic stem cells from bovine blastocysts. Expression of the Pit-Oct-Unc (POU) transcription factor Oct4 was employed to monitor the pluripotent status of cultured cells. No expression of the previously identified bovine Oct4 pseudogene was found, and transcription of the Oct4 ortholog correlated with the proliferative potential of bovine ICM derived cells. Two methods to isolate pluripotent inner cell mass were compared; 90% of trypsin isolated ICMs formed growing cultures, whereas only 12%–23% of the ICMs isolated by immunosurgery attached and grew. Colony formation from complete blastocysts was 55%. The bovine ICM derived cells could be grown for 4–7 passages. However, Oct4 transcripts were only present in the primary cultures, indicating that the initial culture period of bovine ICM derived cells is critical and needs to be optimized to yield true ES cells. In contrast to bovine ICMs, murine ICMs yielded rapidly growing cells, which proliferated for more than 60 passages. Mol. Reprod. Dev. ß 2005 Wiley-Liss, Inc. Key Words: embryonic stem cells; bovine Oct4; pseudogene INTRODUCTION Embryonic stem cells are derived from the inner cell mass of blastocysts and are pluripotent, that is, can differentiate into any cell type in the body including gametes (Weissman, 2000; Rossant, 2001). Application of these cells to study fundamental events in embryonic development, to produce therapeutic delivery systems, and to test drugs in pharmaceutical research has made this a rapidly growing area of research (Lovell-Badge, 2001). Evans and Kaufman (1981) were the first to establish a method for growing stem cells from mouse embryos indefinitely in the laboratory, while Thomson et al. (1998) developed the first human embryonic stem cell line. No true pluripotent ES cells with germ line contribution have been established from mammals other than mouse (Thomson and Marshall, 1998; Niemann and Kues, 2000; Saito et al., 2004; Wolf et al., 2004). Bovine ES-like cells have been reported by ß 2005 WILEY-LISS, INC. several laboratories. The time period over which these cells could be maintained in culture varied from 13 weeks to 3 years and chimera formation has been described with cells from passages 3–10 (Evans et al., 1990; Sims and First, 1993; Stice et al., 1996; Cibelli et al., 1998; Talbot et al., 2000; for review see Gjørret and Maddox-Hyttel, 2005). The correct expression level of Oct4 is considered a key marker for pluripotent cells in the mouse because it is essential for maintenance of an undifferentiated state (Niwa et al., 2000; Pesce and Schöler, 2001). Oct4, a transcription factor of the Pit-Oct-Unc (POU) family, is expressed in pluripotent cells of preimplantation embryos and in all cells of the germ line. In the mouse, Oct4 is present in all embryonic cells up to the morula stage; and becomes restricted to the ICM cells in blastocysts (Nordhoff et al., 2001; Pesce and Schöler, 2001). Orthologous genes have been identified in the genome of humans, primates, and domestic species. Whereas the bovine Oct4 mRNA follows the same pattern of expression as in the mouse during preimplantation development, bovine, and porcine Oct4 proteins are apparently not restricted to the pluripotent ICM cells of blastocysts, but have also been detected in trophectoderm and in all cells of primary blastocyst outgrowths (van Eijk et al., 1999; Kirchhof et al., 2000; Kurosaka et al., 2004). Moreover, van Eijk et al. (1999) isolated a bovine Oct4 pseudogene, which was not known from the mouse system and RT-PCR data suggested that the pseudogene might be expressed in bovine embryos (van Eijk et al., 1999). These latter data compromised the usefulness of Oct4 as a marker for pluripotency in livestock species. The availability of ES cells from farm animals would permit the extension of studies using murine cells, as human embryos and ES cells are constrained by legal and ethical restrictions. A procedure for the isolation of bovine ES cells would facilitate transgenic approaches Grant sponsor: Deutsche Forschungsgemeinschaft (DFG). *Correspondence to: Prof. Heiner Niemann, Department of Biotechnology, Institute of Animal Breeding (FAL), Mariensee, 31535 Neustadt, Germany. E-mail: [email protected] Received 7 February 2005; Accepted 1 June 2005 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mrd.20343 2 P.S. YADAV ET AL. in agriculture and biomedicine (Kues and Niemann, 2004). An important precondition for the isolation of livestock ES cells is the availability of a suitable marker for pluripotency. In the present study, bovine ICM cells were isolated and cultured, and the usefulness of Oct4 as a bovine stem cell marker was assessed. For comparison, murine ES cells were derived from mouse blastocysts and cultured in parallel. MATERIALS AND METHODS Isolation of Feeder Cells A bovine fetus (estimated age 110 days p.c.) was obtained from a commercial slaughterhouse and kept in PBS. The fetus was decapitated, eviscerated, and small (>1 mm3) pieces of sub-dermal tissue were prepared. These tissue explants were placed in microdrops of recalcified bovine plasma and incubated for 1 hr to allow coagulation of the plasma prior to adding culture medium (87% Dulbecco’s Modified Eagles Medium (DMEM, PAA, Cölbe, Germany) and 10% fetal calf serum (batch: 40F2342K, untreated, Gibco, Karlsruhe, Germany), 1% nonessential amino acids (Sigma, Deisenhofer, Germany), 1% vitamin solution (Sigma), 1% antibiotics, 2 mM glutamine, and 0.1 mM mercaptoethanol. Subconfluent cultures were trypsin-EDTA treated and subpassaged for cell multiplication (Kues et al., 2000, 2005). The harvested cells were frozen in aliquots using a mixture of 90% culture medium and 10% dimethylsulfoxid; frozen aliquots were thawed and grown to confluency and then treated with 10 mg/ml mitomycin C (Sigma) for 3 hr. The mitomycin C treated cells were frozen in small aliquots, and seeded 1 day prior usage as feeder cells. Sufficient stocks of the feeder fibroblasts were created and frozen in liquid nitrogen to provide an identical feeder layer for all experiments. In Vitro Production of Bovine Blastocysts Bovine blastocysts were produced in vitro (Eckert and Niemann, 1995; Holm et al., 1999; Wrenzycki et al., 2001). Briefly, ovaries were collected from a local slaughterhouse and transported to the laboratory in an insulated container at 25–308C in PBS. Oocytes were isolated by slicing the ovaries in PBS containing heparin and 1% BSA. The oocytes were matured in TCM 199 (Sigma) containing 0.2 mM L-glutamine and 25 mM HEPES. For oocyte maturation, TCM199 containing 0.1% fatty acid free BSA (Sigma, A7030) medium was supplemented with 10 IU/ml eCG and 5 IU hCG/ml (Suigonan, Intervet, Germany) for 24 hr in a CO2 incubator. Cumulus oocyte complexes (COC) were cultured in groups of 15–20 in 100 ml maturation drops under silicone oil in a humidified atmosphere composed of 5% CO2 in air at 398C for 24 hr. Matured oocytes were rinsed in fertilization medium (Fert-TALP supplemented with 6 mg/ml BSA) and fertilized in Fert-TALP containing 10 mM hypotaurine (Sigma), 1 mM epinephrine (Sigma), 0.1 IU/ml heparin (Serva, Heidelberg, Germany), and 6 mg/ml BSA. Frozen thawed semen from bulls of proven fertility was used for IVF. The final sperm concentration added per drop of 100 ml containing 10–15 oocytes was 1 106 sperm cells/ml. Fertilization took place over 18 hr co-culture of sperm and oocytes. Presumptive zygotes were cultured in 30 ml of synthetic oviduct fluid (SOF) supplemented with 1% BSA. Embryos were cultured in mixture of 5% CO2, 7% O2, 88% N2 (Air Products, GmbH Hattingen, Germany) in modular incubator chambers (615300 ICN Biomedical, Inc., Aurora OH). Expanded blastocysts were harvested on day 8 of development (day 0 ¼ IVF). Isolation of Inner Cell Masses From Blastocysts The inner cell masses (ICM) were isolated either by trypsin treatment or by immunosurgery. In both cases, the zona pellucida was first removed by incubation in 1.0% pronase (Sigma, P6911) in PBS (w/v) until it dissolved. For trypsin isolation, zona free blastocysts were transferred into 0.25% trypsin-EDTA solution and observed under a microscope until the trophectoderm cells became loose, and were shed from the ICM by pipetting. These ICMs were transferred into PBS and then seeded on feeder cells. For a modified immunosurgical isolation of ICMs (Solter and Knowles, 1975), the zona-free blastocysts were washed three times in PBS with 1% polyvinyalcohol (PVA) (Sigma P 8136) and placed in 10 mM trinitrobenzene-sulphonic acid (TNBS) (Sigma P 2297) in PBS containing 3 mg/ml polyvinylpyrrolidone for 10 min. After washing in 1% PBS-PVA, they were treated with rabbit anti dinitrophenyl-BSA (anti DNP BSA 61-006 ICN Biochemicals, Eschwege, Germany) diluted 3:7 in PBS for 10 min. The anti-DNPBSA crossreacts with trinitrophenol groups. Outer trophectodermal cells were then lysed with guinea pig complement serum (Sigma S1639, 1:4) for 15 min. The isolated ICMs were washed with PBS-PVA and then seeded on feeder layers. The culture medium for bovine embryonic cells consisted of 77% DMEM, 20% FCS, 1% nonessential amino acids, 1% vitamin solution, and 1% penicillin-streptomycin solution. In some experiments, the culture medium was supplemented with either LIF (40–80 ng/ml), hIGF (82 ng/ml), TNF-b (0.12 ng/ml), and bFGF (4 ng/ml). Culture media were changed every second day. Suspension cultures were maintained in hanging drops. Hanging drops were prepared by placing drops of 30 ml media on the inner surface of the cover of 6-well plates. The wells of the 6-well plates were filled with PBS to prevent drying of drops. Alkaline Phosphatase Staining Expression of alkaline phosphatase (AP) was analyzed in blastocysts, ICMs, and cultured cells. The specimens were washed twice with PBS before fixing them in 3.7% paraformaldehyde for 10 min. After washing with PBS, they were incubated in AP staining solution (25 mM Tris HCl pH 9.0, 150 mM NaCl, 4 mM MgCl2; 0.4 mg/ml sodium naphthylphosphate; 1.0 mg/ ml fast red) for 1 hr (Kues et al., 2000). AP positive stained cells appeared red, while negative cells were colorless or brownish. STEM CELLS FROM BOVINE AND MURINE BLASTOCYSTS RT-PCR Amplification of Selected Transcripts Poly(A)þ RNA from groups of 5–10 blastocysts was isolated using a Dynabeads mRNA DIRECT Kit (Dynal, Norway, Product number 610.11). Nearly confluent ICM derived cells in 6-well culture dishes at the 2nd, 3rd, and 4th passage (or 24-well dishes with primary colonies) were washed twice with PBS, and then stored at 808C prior to isolation of total RNA. Isolation of RNA from cells was accomplished with Trizol (1 ml/6-well culture plate) (Trizol Invitrogen Cat No. 15596026). The Trizol extracts were transferred to 1.5 ml safe lock tubes and 200 ml of chloroform were added, the aqueous and organic phase were mixed by shaking prior to centrifugation for 15 min at 9,700g. The supernatant was collected and 0.5 ml isopropylalcohol was added per ml Trizol. The samples were then centrifuged for 15 min, the supernatant was removed and the RNA pellet was washed with 1 ml 70% cold ethanol. The pellet was air dried for several minutes and dissolved in 60 ml RNase free water. The RNA was treated with RNase-free DNase for removal of DNA, then the RNA concentration was measured using a Nanodrop spectrophotometer (ND-1000 spectrophotometer, Rockland, MD). The mRNAs were reverse transcribed in a reaction mixture consisting of 1 RT buffer (50 mM KCl, 10 mM Tris HCl pH 8.3, Perkin Elmer), 5 mM MgCl2, 1 mM each of dNTP (Amersham, Brunswick, Germany) 20 IU RNase inhibitor (Perkin Elmer), and 50 U reverse transriptase (RT, Perkin Elmer) in a total volume of 20 ml using 1.0 ml of 50 mM random hexamer primers (Perkin-Elmer, Vaterstetten, Germany). The RT reaction was carried out at 258C for 10 min and then 428C for 1 hr followed by denaturation at 998C for 5 min. The polymerase chain reaction was performed with the reverse transcribed product using one or two embryo equivalents or 10% of the RT-reaction from cultured cells. The final volume of the PCR reaction mixture was 50 ml (1 PCR Buffer, 20 mM Tris buffer HCl pH 8.4, 1.5 mM MgCl2, 200 mM dNTPs, 0.25 mM of each primer). RT-PCR products were separated by electrophoresis on a 2% agarose gel in 1 TBE buffer (90 mM Tris, 90 mM borate, 2 mM EDTA, pH 8.3 containing 0.2 mg/ml ethidium bromide). An image of each gel was recorded using a CCD camera (Quantix, Photometrics, Munich Germany). Details of primers are given in Table 1. The amplicon of the control poly(A) polymerase gene does not contain an intron, therefore the PCR products using RNA or genomic DNA as templates are identical. For sequencing, the DNA bands were cut out under long wave length UV illumination (365 nm), purified using a DNA gel band purification kit (Amersham Biosciences, Freiburg, Germany) and sequenced with the Oct-26 primer (van Eijk et al., 1999). Isolation and Culture of Mouse Embryo Derived Cells Mouse blastocysts were collected by flushing the uteri of superovulated wildtype or Oct4-GFP transgenic (OG2) mice at day 3.5 after mating. Oct4 positive transgenic blastocysts were used for culture or ICM isolation. ICMs were isolated and cultured as described for bovine embryos. RESULTS Expression of the Bovine Oct4 Gene but not of the Pseudogene in Bovine Blastocysts Expression of Oct4 was assessed in bovine embryos and somatic tissues by RT-PCR with splice-site spanning primers (Fig. 1A). To determine if the obtained RT-PCR fragments resulted from amplification of the Oct4 ortholog or the Oct4 pseudogene, PCR products were isolated and directly sequenced. The sequences of the Oct4 ortholog (AF022987) and the Oct4 pseudogene (AF022988, van Eijk et al., 1999) differ by nucleotide exchanges at positions 754 and 1,245 (numbering according to AF022987). The obtained sequences from bovine blastocysts revealed that blastocysts exclusively expressed the bovine Oct4 ortholog, whereas control PCRs with bovine genomic DNA isolated from white blood cells (Fig. 1B), liver, and kidney amplified exclusively the pseudogene. No amplification of Oct4 fragments was obtained from mRNAs isolated from white blood cells, liver, and kidney (Fig. 1A). Thus the presence of a bovine Oct4 pseudogene could be verified, however, no evidence of pseudogene transcription was found. Together with recent data indicating that Oct4 mRNA expression patterns are conserved between mammalian species (Kurosaka et al., 2004), this suggests that Oct4 transcription can indeed be used as a marker for pluripotent cells in cattle. TABLE 1. Details of Primers Used for Identification of Bovine Oct-4 Expression Name of primer Bovine Oct4 PolyA polymerase Mouse Oct4 3 Primer sequence Upper 50 -GTT CTC TTT GGA AAG GTG TTC Lower 50 -ACA CTC GGA CCA CGT CTT TC Uppper 50 -CCT CAC TGC ACA ATA TGC CGT CTT CAC CTG Lower 50 -AGC CAA TCC ACT GTT CTC CCA CCT TTA CTC Upper 50 -GAA CAG TTT GCC AAG CTG CTG Lower 50 -CCG GTT ACA GAA CCATAC TCG Gene bank accession number Annealing temp. (PCR cycles) AF022987 57 (40) X 63436 54 (36) X 52437 54 (40) Reference Van Eijk et al., 1999 Wrenzycki et al., 2001 Schöler, 1991 4 P.S. YADAV ET AL. Fig. 1. Oct4 but not its pseudogene is expressed in bovine blastocysts. A: Lane 1, RT-PCR amplification of Oct4 from bovine blastocysts; lane 2 and 3, controls without RT and without template; lanes 4, 6, 8, and 10, RT-PCR of total RNA from bovine white blood cells, liver, kidney, and lung; lane 11, PCR Oct4-fragment from genomic DNA; lanes 5, 7, 9 controls without RT; and lane 12 without template. Control reactions with primers specific for poly(A) polymerase are shown below. B: The amplified Oct4 products obtained from blastocyst mRNAs and genomic DNA were isolated (indicated by white boxes in A) and sequenced in pools. Short stretches of the sequences containing the regions of nucleotide mismatches (boxed nt positions) between the Oct4 gene and its pseudogene are shown, the numbering is according to GenBank accession no AF022987. The sequences derived from blastocysts match 100% with the Oct4 gene. Both nucleotide exchanges of the described pseudogene were present in genomic DNA at positions 1,245 and 754. For sequence reactions, the reverse Oct-26 primer was used, therefore, the noncoding strand is shown. C: Control sequences of bovine Prnp genes of cattle with known single nucleotide polymorphisms (SNP). Arrows indicate the SNP positions where signal peaks for both allele variants are present. Isolation and Initial Cultures of Bovine ICM Derived Cells ICMs, the removal of trophectodermal cells was not complete. The remaining trophectodermal cells produced blastocyst-like structures with diameters of up to 500–600 mm before attaching to the feeder cells. Primary colonies formed after 2 weeks and grew as a single sheet of cells (Fig. 2B). These sheet-like colonies were physically separated into small pieces for subpassaging since trypsin treatment was not effective. In subcultures, the pieces increased in size before attaching (Fig. 2C). The typical time for each passage was 10– 12 days. It was found that 55% of the intact blastocysts In the first experiment, 20 intact blastocysts and 50 ICMs isolated by trypsin treatment were cultured on feeder cells. Intact blastocysts hatched prior to attachment, and the trypsin isolated ICMs regained a blastocyst-like shape due to outgrowth of the remaining trophectodermal cells (Fig. 2A). Different trypsin treatment times and different enzyme concentrations were tested. Under conditions, which allowed the survival of STEM CELLS FROM BOVINE AND MURINE BLASTOCYSTS 5 Fig. 2. Different growth phenotypes of bovine ICM derived cultures. A–F: Bovine ICMs and initial cultures. (A) Reexpanded bovine trypsinisolated ICM before attachment (40), (B) Trypsin isolated ICM, passage 2, sheet like colony (40), (C) Passage 2 colony after physical detachment (40), (D) Donut shaped growth in LIF supplemented medium (40), (E) Bovine ICM derived by immunosurgery, attached on feeder cells (200), (F) Primary colony growth from immunoisolated ICM. G–H: Murine ICMs and derived cell cultures: (G) Murine trypsin isolated ICM, day 13 of attachment (40), (H) Proliferating ICM derived cells at passage 6. formed primary colonies, which could be passaged 4– 5 times. By comparison, 90% of the cells isolated from trypsin digested ICMs formed primary colonies which could be passaged 6–7 times over a period of 60–65 days (Table 2) before proliferation ceased. When trypsin isolated ICMs were cultured in medium with LIF (40 ng/ml) supplementation, they grew into donut shaped structures (Fig. 2D). The size of the donut-like structure increased to >1 mm and cells of two different morphologies became visible. Round TABLE 2. Comparison of Culture Behavior of Embryo Derived Cells From Intact Bovine Blastocysts and Trypsin-EDTA Isolated ICMs Starting material Blastocysts ICMs (trypsin isolated) a No. of Embryos Primary colony formation (%) Passagesa 20 50 11 (55%) 45 (90%) >4 >7 Sub-passaging was done when the cells reached 50%–70% confluency. 6 P.S. YADAV ET AL. individual cells were present on the bottom of the structure, while the surface was composed of tightly bound cells. Immunosurgically Isolated ICMs In the second experiment, bovine ICMs were isolated by immunosurgery and cultured in DMEM/20% FCS supplemented with (1) LIF (40 ng/ml), hIGF(2 ng/ml), TNF-ß (0.12 ng/ml), and bFGF (4 ng/ml); (2) LIF (40 ng/ ml); (3) LIF (80 ng/ml); or (4) without growth factor supplement. The rate of primary colony formation and the proportion of proliferating colonies are presented in Table 3. Immunosurgically isolated ICMs attached within 3– 4 days after seeding on the feeder layer in all tested media (Fig. 2E). The initial colonies remained very small for 1 week, but after 2 weeks in culture, large primary colonies could be observed (Fig. 2F). In the group grown in medium containing all four growth factors, 11.5% of the seeded ICMs produced primary colonies. The group supplemented with LIF (40 ng/ml) had the highest percentage of colony formation (21.5%). The morphology of cells in these colonies was different from that of enzyme digested ICMs or intact blastocyst colonies. Trypsin could be used to detach these colonies and the cultures were passaged after gentle physical separation. Small clumps of cells were better at attachment and growth after passaging than individual cells. Most cells proliferated well in the first four passages and then grew at a slower rate up to the 6th passage, finally arresting after 50–60 days. RT-PCR analysis revealed that only the initial cultures expressed Oct4 mRNA, from the second passage Oct4 mRNA was no longer detectable (Fig. 3A,B). The average passage time was 10–14 days. In hanging drops, the floating cells did not proliferate. Permanent Culture of Murine ICM-derived Cells Day 3.5 p.c. murine blastocysts from wildtype and Oct4-EGFP (OG2) transgenic mice served as controls to validate the ICM isolation protocols and culture conditions. Results were identical for transgenic and nontransgenic embryos, and ICMs (Table 4). Attachment rates and primary colony formation for trypsin isolated murine ICMs were higher than for intact blastocysts. GFP expression was observed in cells from the OG2transgenic embryos, but started to diminish from day 4 of culture. In unattached blastocysts, GFP was visible for up to 10 days in culture. Primary colonies formed after 10–13 days of culture starting with intact embryos or ICMs (Fig. 2G). Primary colonies in 24-well plates were transferred to 6-well plates and then to 25 cm flasks for amplification (Fig. 2H). ICM derived cells from three different batches survived for 62, 58, 57 passages, respectively and could be maintained in culture for more than 225 days (Table 5). In hanging drops, the cells formed loose embryoid bodies within 1 week. Without a feeder cell layer, embryonic cells could attach to the plastic but growth was slow. Murine ICM derived cells were negative for AP staining, but expressed Oct4 mRNA at passages 41 and 46 (Fig. 3C). DISCUSSION The transcription factor Oct4 is essential for the germ cell lineage in the mouse where it is required at critical levels to maintain embryonic stem cells in a pluripotent state (Niwa et al., 2000; Buehr et al., 2003). The bovine Oct4 shares high sequence homology with its mouse, primate, and human orthologs (Nordhoff et al., 2001; Mitalipova et al., 2003), and has 90.6% and 81.7% overall identity at the protein level to human and murine orthologs, respectively. However, in bovine cells, the usefulness of Oct4 has been questioned by the identification of a bovine Oct4 pseudogene and suggestions that this pseudogene might be transcribed in bovine embryos (van Eijk et al., 1999). The apparent discrepancy to the data presented here might be due to the fact that van Eijk et al. (1999) used an indirect method to discriminate between the pseudogene and Oct4 mRNA. They employed a mutagenised PCR primer to introduce a restriction site in the Oct4 amplicon, followed by EagI digest and gel analysis. In the present study, Oct4 expression was detected by RT-PCR with completely matching primers. Direct sequencing of the PCR product allowed unequivocal discrimination between the Oct4 ortholog and pseudogene, as they differ by nucleotide exchanges at positions 754 and 1,245. Our sequence data revealed that bovine blastocysts expressed exclusively the Oct4 ortholog; expression of the pseudogene could not be detected in embryonic or adult tissues such as liver, kidney, and white blood cells. In contrast, control experiments with genomic DNA from liver, kidney, and white blood cells, resulted in successful amplification of the pseudogene, confirming presence of the pseudogene in the bovine genome and demonstrating that the PCR conditions are suitable and sensitive for its detection. Recently, it was TABLE 3. Proliferation of Bovine Immunosurgically Isolated ICMs in the Presence of Various Growth Factors Media supplements LIF, IGF, TNFb, bFGF LIF (40 ng/ml) LIF (80 ng/ml) None No. of trials No. of ICMs No. of primary colonies % primary colonies No. of passages Days in culture 11 13 3 12 78 87 33 79 9 19 6 13 11.5 21.8 18.2 16.4 4–6 4–6 3 4–6 65 57 30 56 STEM CELLS FROM BOVINE AND MURINE BLASTOCYSTS 7 Fig. 3. Expression of Oct4 in ICM derived cell cultures. A: Oct4 is expressed in passage 1 bovine ICM cultures. M, 100 bp ladder; lane 1, passage 1 bovine ICM cells (day 14 of culture); lane 2 and 3, controls without RT or template; lane 4, Oct4 PCR with bovine genomic DNA; lane 5, control without template. B: RT-PCR of Oct4 from passages 2–4. M, 100 bp ladder, lanes 1, 3, 5 passage 2–4 bovine ICM cells; lanes 2, 4, 6, controls without RT; lane 7, control without template; lane 8, Oct4 PCR with bovine genomic DNA; lane 9, control without template. C: RTPCR of Oct4 from murine ICM derived cultures employing mouse specific primers. M, 100 bp ladder, lanes 1 and 2, Oct4 RT-PCR from passages 41 and 47, lane 3, control without RT; lane 4, control without template. In parallel, control RT-PCRs with poly(A) polymerasespecific primers were performed (bottoms). shown that the Oct4 mRNA pattern is restricted to ICM cells in bovine blastocysts as in mice (Kurosaka et al., 2004). The more widespread distribution of the Oct4 protein in ICM and trophectodermal cells of bovine blastocysts (van Eijk et al., 1999; Kirchhof et al., 2000) possibly reflects the prolonged bovine preimplantation phase compared to that of the mouse. Recent data show that the Oct4 protein becomes restricted to the embryonic disc of hatched day 12 bovine embryos which confirms this view (Gjørret and Maddox-Hyttel, 2005). Taken together, these observations make Oct4 transcription a suitable marker for the identification of pluripotent cells in cattle. During culture of bovine ICMs, expression of Oct4 mRNA was rapidly lost after the first passage, which correlates well with their limited capacity for self renewal and proliferation. AP activity has previously been used to identify pluripotent cells in cultures from livestock species (Talbot et al., 2000, 1993; Li et al., 2003). In the present study, intact bovine and murine blastocysts were positive for AP activity. However, we found that AP activity was weak or absent in isolated bovine ICMs and in bovine ICM derived cell cultures. In some colonies of later passages, a few AP positive cells could be identified. A similar heterogeneous staining pattern of AP activity in bovine ICM derived cells had been reported previously (van Stekelenburg-Hamers et al., 1995; Gjørret and Maddox-Hyttel, 2005). These observations suggest TABLE 4. Murine Embryo Derived Cells: Efficiency of Isolation and Development Starting material No. No. of attached (%) Primary Colony (%) Transgenic blastocysts Nontransgenic blastocysts Transgenic ICM Nontransgenic ICM 12 10 24 16 3 (25%) 6 (60%) 17 (91%) 16 (100) 3 (25%) 4 (40%) 17 (91%) 14 (87%) 8 P.S. YADAV ET AL. TABLE 5. Maintenance of Murine Cells in Culture on Bovine Feeders Cultures 1 2 3 Number of days Number of passages Av. time/passage (days) 234 227 220 62 57 58 3.8 4.0 3.8 that AP expression is not a robust marker for pluripotency in bovine ICM derived cells. In the present study, two different methods, trypsin digestion and immunosurgery, were tested for their suitability for ICM isolation from bovine blastocysts. Trypsin digestion had previously been shown to effectively remove porcine trophectoderm cells (Li et al., 2003). In the present study, murine and bovine ICMs isolated with trypsin showed an increased attachment rate and improved primary colony formation as compared to intact blastocysts. This substantiates recent findings for porcine embryos (Li et al., 2003). The trypsin digestion method yielded a population of murine cells which could be continuously cultured, while the bovine system resulted in sheet shaped colonies, in which the cells had the appearance of trophectoderm, suggesting that lysis of trophectodermal cells was incomplete. Trypsin has been reported to be ineffective for detaching primary ES cell colonies for initial passaging (Stice et al., 1996; Talbot et al., 2000), and such cultures are commonly passaged by physical dissociation. In the present study, primary colonies derived from trypsin digested ICMs, contained cells of two distinct morphologies. Individual, round cells were only found in the central parts of the colonies. Immunosurgery is the conventional method for isolating ICMs from blastocysts (Thomson and Marshall, 1998). The morphology of the cells obtained in the present study after immunosurgery was quite different from that of cells derived from trypsin digestion in that individual cells could be easily distinguished by phase contrast microscopy. Four different culture media were used after immunosurgical isolation of ICMs to explore the effect of growth factors. For immunosurgically isolated ICMs, growth factors caused variation in attachment and growth, but only LIF seemed to stimulate colony formation. However, one has to bear in mind that the medium also contained fetal calf serum, which may have masked effects of individual growth factors. When all growth factors were used together, this resulted in low-primary colony formation rates, showing that the mixture of growth factors including LIF did not improve the growth of cells from the ICMs. The morphology of cells after growth factor supplementation with or without LIF remained similar. Recently, a synergistic effect between bone morphogenetic protein 4 (BMP4) and LIF was found in maintaining the self renewal of murine ES cells (Qi et al., 2004). This makes BMP4 a good candidate molecule for testing on bovine ICM derived cells. The culture of murine and human stem cells usually requires the presence of feeder cells (Thomson and Marshall, 1998; Richards et al., 2002; Amit et al., 2004). Feeder cells presumably produce growth factors and other substances that support proliferation and prevent the differentiation of pluripotent cultured cells (Anderson, 1992). Livestock ES-like cells are also commonly cultured on feeder cells because the molecular pathways and key molecules required to maintain pluripotency in these species are unknown (Wolf et al., 2004). In the present study, mitomycin-treated bovine fetal fibroblasts (BFF) from explant cultures (Kues et al., 2005) were used as a feeder layer. These feeder cells supported the attachment of intact embryos, ICMs and the proliferation of bovine ICM cell colony formation for 6–7 passages and murine ICM cell proliferation for more than 60 passages. Previously, murine embryonic fibroblasts and STO cells or bovine epithelial layers were used as feeders for bovine ICM cells (Saito et al., 1992; van Stekelenburg-Hamers et al., 1995). Successful use of BFF cells to culture murine ICM cells indicates that BFF cells could potentially be used as feeder cells for homologous as well as heterologous ES cells. In contrast to the limited proliferation of bovine ICM derived cells, cells from murine blastocysts could be grown well for more than 200 days over 60 passages on the same feeder layers and under identical culture conditions. In the mouse, ES cells appear to arise from the epiblast cells (Brook and Gardner, 1997); however, attempts to isolate ES cells from bovine embryonic discs of day 12 embryos did not result in permanent cell lines (Gjørret and Maddox-Hyttel, 2005). AP activity in murine ES cells was negative but the cells expressed Oct4 in the 41th and 46st passage. The cells formed embryoid bodies in suspension culture and differentiated into various cell types when cultured without feeder cells. In summary, bovine embryonic fibroblast cells can be used as feeder cells for both bovine and murine ICM cells. However, bovine ICM derived cells proliferated for a limited period. 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Progress with nonhuman primate embryonic stem cells. Biol Reprod 71:1766–1771. Wrenzycki C, Herrmann D, Keskintepe L, Martins A , Jr., Sirisathien S, Brackett B, Niemann H. 2001. Effects of culture system and protein supplementation on mRNA expression in pre-implantation bovine embryos. Hum Reprod 16:893–901. Rev. sci. tech. Off. int. Epiz., 2005, 24 (1), 285-298 Transgenic farm animals: present and future H. Niemann, W. Kues & J.W. Carnwath Department of Biotechnology, Institute for Animal Breeding (FAL), Mariensee, 31535 Neustadt, Germany Summary Until recently, pronuclear microinjection of deoxyribonucleic acid (DNA) was the standard method for producing transgenic animals. This technique is now being replaced by more efficient protocols based on somatic nuclear transfer that also permit targeted genetic modifications. Lentiviral vectors and small interfering ribonucleic acid technology are also becoming important tools for transgenesis. Transgenic farm animals are important in human medicine as sources of biologically active proteins, as donors in xenotransplantation, and for research in cell and gene therapy. Typical agricultural applications include improved carcass composition, lactational performance and wool production, as well as enhanced disease resistance and reduced environmental impact. Product safety can be ensured by standardisation of procedures and monitored by polymerase chain reaction and array technology. As sequence information and genomic maps of farm animals are refined, it becomes increasingly practical to remove or modify individual genes. This approach to animal breeding will be instrumental in meeting global challenges in agricultural production in the future. Keywords Agricultural application – Environmental benefit – Farm animal – Gene pharming – Lentiviral vector – Microinjection – Nuclear transfer – Small interfering ribonucleic acid – Transgenic – Xenotransplantation. Introduction: evolution of transgenic technologies The first transgenic livestock were produced almost 20 years ago by microinjection of foreign deoxyribonucleic acid (DNA) into the pronuclei of zygotes (33). However, as microinjection has several significant shortcomings – including low efficiency, random integration and variable expression patterns related to the site of integration – research has focused on alternative methodologies for improving efficiency of generating transgenic livestock (Table I). These methodologies include: – sperm-mediated DNA transfer (12, 54, 55) – intracytoplasmic injection of sperm heads carrying foreign DNA (71, 72) – injection or infection of oocytes and/or embryos by different types of viral vectors (11, 35, 37) – ribonucleic acid (RNA) interference technology (small interfering RNA: siRNA) (14) – the use of nuclear transfer (5, 13, 21, 53, 86). To date, somatic nuclear transfer, which has been successful in ten species – albeit at low efficiency (47, 103) – holds the greatest promise for significant improvements in the generation of transgenic livestock (Table I). Further qualitative improvements may be derived from technologies that allow precise modifications of the genome, including targeted chromosomal integration by site-specific DNA recombinases, such as Cre or flippase (FLP), or methods that allow temporally and/or spatially controlled transgene expression (9, 45). The first genomes of farm animals (cattle, chicken) were sequenced and annotated in 2004 (2, 3). Thus, after approximately 7,000 years of domestic animal selection (16) based on the random mutations caused by radiation and oxidative injury to the genome, technology is now available to introduce or remove known genes with known functions. Despite the inherent inefficiency of microinjection technology, a broad spectrum of genetically modified large animals has been generated for applications in agriculture and biomedicine (Table II). The use of transgenic livestock for ‘gene pharming’ has now reached the level of commercial exploitation (47). This paper provides a brief summary of the current status of transgenic animal production and look at future implications. The authors 286 Rev. sci. tech. Off. int. Epiz., 24 (1) focus on large domesticated species and do not cover the recent developments in poultry breeding or aquaculture. limitations of conventional and recombinant production systems for pharmaceutical proteins (63, 82) and has advanced to the stage of commercial application (27, 107). The mammary gland is the preferred production site, mainly because of the quantities of protein that can be produced in this organ using mammary gland-specific promoter elements and established methods for extraction and purification of that protein (63, 82). Biomedical applications of transgenic domestic animals Pharmaceutical production in the mammary gland of transgenic animals Guidelines developed by the Food and Drug Administration (FDA) in the United States of America (USA) require monitoring of the animals’ health, sequence validation of the gene construct, characterisation of the isolated recombinant protein, and monitoring of the genetic stability of the Gene ‘pharming’ entails the production of recombinant biologically active human proteins in the mammary glands of transgenic animals. This technology overcomes the Table I Evaluation of gene transfer methodology* Methodology Animal usage Site of integration Integration prescreening Homologous recombination Multiple transgenes Maximum construct size limitation Founder Technical mosaicism difficulty Microinjection High Random No No Inefficient Artificial chromosomes Yes Somatic nuclear transfer Low Prescreened Yes Possible Yes Transfection limited/ artificial chromosomes No Implantation of transgenic spermatogonia Low Random Yes Not yet Not yet Transfection limited No Lentivirus injection Low Random No No Yes ~ 5 kb Yes Sperm-mediated gene transfer method Low Random No No Inefficient Not known Not known * all grading is based on the fully matured technology Table II Current status of transgenesis in livestock Area of application Biomedicine Gene pharming Antibody production Xenotransplantation Solid organs Cells/tissues Blood replacement Disease model Agriculture Carcass composition Leaner meat Healthful lipids Lactational performance Environmental impact Wool production Disease resistance Fertility Early experimental phase Experimental stage Advanced experimental stage Transition experiments/ practical use Practical use 287 Rev. sci. tech. Off. int. Epiz., 24 (1) transgenic animals over several generations. This has necessitated, for example, the use of animals from scrapiefree countries (New Zealand) and the maintenance of production animals under strict hygienic conditions. Products derived from the mammary gland of transgenic goats and sheep, such as antithrombin III (ATIII), α-antitrypsin or tissue plasminogen activator (tPA), have progressed to advanced clinical trials (47). Phase III trials for a recombinant human ATIII product have been completed and an application has been filed for European Market Authorisation. The protein is expected to be registered and on the market by the end of 2005. The product is employed for the treatment of heparin-resistant patients undergoing cardiopulmonary bypass procedures. At the same company that manufactures this product, 11 transgenic proteins have been expressed in the mammary gland of transgenic goats at more than one gram per litre. The enzyme α-glucosidase from the milk of transgenic rabbits has orphan drug status and has been successfully used for the treatment of Pompe’s disease (94) (in the USA the term ‘orphan drug’ refers to a product that treats a rare disease affecting fewer than 200,000 Americans). Similarly, recombinant C1 inhibitor produced in the milk of transgenic rabbits has completed phase III trials and is expected to receive registration within the next 24 months. A topical antibiotic against Streptococcus mutans, for prevention and treatment of dental caries, has completed phase III trials and should be launched shortly. The global market for recombinant proteins from domestic animals is expected to exceed US$1 billion in 2008 and to reach US$18.6 billion in 2013. Some gene constructs have failed to produce economically significant amounts in the milk of transgenic animals, indicating that the technology needs further refinements to achieve high-level expression. This is particularly true for genes that have complex regulation, such as those coding for erythropoietin or human clotting factor VIII (36, 41, 62, 67). With the advent of transgenic crops that produce pharmacologically active proteins, there is now an array of recombinant technologies that will allow selection of the most appropriate production system for each required protein (58). The production of edible vaccines in transgenic crop plants against, for example, foot and mouth disease, might become an important application for animal health (102). Antibody production in transgenic animals Numerous monoclonal antibodies are being produced in the mammary gland of transgenic goats (63). Cloned transgenic cattle produce a recombinant bispecific antibody in their blood (31). Purified from serum, the antibody is stable and mediates target cell-restricted T cell stimulation and tumour cell killing. An interesting new development is the generation of trans-chromosomal animals. A human artificial chromosome containing the complete sequences of the human immunoglobulin heavy and light chain loci was introduced into bovine fibroblasts, which were then used in nuclear transfer. Transchromosomal bovine offspring were obtained that expressed human immunoglobulin in their blood. This system could be a significant step forward in the production of human therapeutic polyclonal antibodies (51). Further studies will show whether the additional chromosome will be maintained over future generations and how stable expression will be. Blood replacement Functional human haemoglobin has been produced in transgenic swine. The transgenic protein could be purified from the porcine blood and showed oxygen-binding characteristics similar to natural human haemoglobin. The main obstacle was that only a small proportion of porcine red blood cells contained the human form of haemoglobin (90). Alternative approaches to produce human blood substitutes have focused on the chemical cross-linking of haemoglobin to the superoxide-dismutase system (20). Xenotransplantation of porcine organs to human patients Today more than 250,000 people are alive only because of the successful transplantation of an appropriate human organ (allotransplantation). However, progress in organ transplantation technology has led to an acute shortage of appropriate organs, and cadaveric or live organ donation does not meet the demand in Western societies. To close the growing gap between demand and availability of appropriate human organs, porcine xenografts from domesticated pigs are considered to be the best alternative (47, 77). Essential prerequisites for successful xenotransplantation are: – overcoming the immunological hurdles – preventing the transmission of pathogens from the donor animal to the human recipient – ensuring the compatibility of the donor organs with human anatomy and physiology. The major immunological obstacles in porcine-to-human xenotransplantation are: – hyperacute rejection (HAR) – acute vascular rejection (AVR) – cellular rejection, and eventually – chronic rejection (101). 288 Hyperacute rejection occurs within seconds or minutes, when, in the case of a discordant organ (e.g. in transplanting from pig to human), pre-existing antibodies react with antigenic structures on the surface of the porcine cells and activate the complement cascade; in other words, the antigen-antibody complex triggers formation of the membrane attack complex. Induced xenoreactive antibodies are thought to be responsible for AVR, which occurs within days after transplantation of a xenograft; disseminated intravascular coagulation (DIC) is a predominant feature of AVR (17, 52). Human thrombomodulin and heme-oxygenase 1 are crucially involved in the etiology of DIC and are targets for future transgenic modifications to improve the long-term survival of porcine xenografts by creating multi-transgenic pigs. When using a discordant donor species such as the pig, overcoming HAR and AVR are the pre-eminent goals. Two main strategies have been successfully explored for long-term suppression of HAR: synthesis of human regulators of complement activity (RCAs) in transgenic pigs (18, 77) and the knockout of α-gal epitopes, the antigenic structures on the surface of the porcine cells that cause HAR (52, 74, 105). Survival rates, after the transplantation of porcine hearts or kidneys expressing transgenic RCA proteins to immunosuppressed nonhuman primates, reached 23 to 135 days, indicating that HAR can be overcome in a clinically acceptable manner (4). The successful xenotransplantation of porcine organs with a knockout of the 1,3-α-galactosyltransferase gene, eliminating the 1,3-α-gal-epitopes produced by the 1,3-α-galactosyltransferase enzyme, has recently been demonstrated. Upon transplantation of porcine hearts or kidneys from these α-gal-knockout pigs to baboons, survival rates reached two to six months (52, 105). A particularly promising strategy to enhance long-term graft tolerance is the induction of permanent chimerism via intraportal injection of embryonic stem (ES) cells (28) or the co-transplantation of vascularised thymic tissue (105). Recent findings have revealed that the risk of porcine endogenous retrovirus transmission is negligible and show that this critical danger could be eliminated, paving the way for preclinical testing of xenografts (47). Despite further challenges, appropriate lines of transgenic pigs are likely to be available as organ donors within the next five to ten years. Guidelines for the clinical application of porcine xenotransplants are already available in the USA and are currently being developed in other countries. The general consensus of a worldwide debate is that the technology is ethically acceptable provided that the individual’s well-being does not compromise public health. Economically, xenotransplantation will be viable, as the enormous costs of maintaining patients suffering from severe kidney disease using dialysis or supporting those suffering from chronic heart disease could be reduced by a functional kidney or heart xenograft. Rev. sci. tech. Off. int. Epiz., 24 (1) Farm animals as models for human diseases Mouse physiology, anatomy and life span differ significantly from those of humans, making the rodent model inappropriate for many human diseases. Farm animals, such as pigs, sheep or even cattle, may be more appropriate models in which to study potential therapies for human diseases that require longer observation periods than those possible in mice, e.g. atherosclerosis, non-insulin-dependent diabetes, cystic fibrosis, cancer and neuro-degenerative disorders (34, 56, 70, 92). Cardiovascular disease is an increasing health problem in aging Western societies, where coronary artery diseases account for the majority of deaths. Because genetically modified mice do not manifest myocardial infarction or stroke as a result of atherosclerosis, new animal models, such as swine that exhibit these pathologies, are needed to develop effective therapeutic strategies (32, 80). An important porcine model has been developed for the rare human eye disease retinitis pigmentosa (PR) (73). Patients with PR suffer from night blindness early in life, a condition attributed to a loss of photoreceptors. Transgenic pigs that express a mutated rhodopsin gene show great similarity to the human phenotype and effective treatments are being developed (61). The pig could be a useful model for studying defects of growth-hormone releasing hormone (GHRH), which are implicated in a variety of conditions such as Turner syndrome, hypochrondroplasia, Crohn’s disease, intrauterine growth retardation or renal insufficiency. Application of recombinant GHRH and its myogenic expression has been shown to alleviate these problems in a porcine model (26). Development of further ovine and porcine models of human diseases is underway (29). An important aspect of nuclear-transfer-derived large animal models for human diseases and the development of regenerative therapies is that somatic cloning per se does not result in shortening of the telomeres and thus does not necessarily lead to premature ageing (85). Telomeres are highly repetitive DNA sequences at the ends of the chromosomes that are crucial for their structural integrity and function and are thought to be related to lifespan. Telomere shortening is usually correlated with severe limitation of the regenerative capacity of cells, the onset of cancer, ageing and chronic disease with significant impacts on human lifespans (85). Expression of the enzyme telomerase, which is primarily responsible for the formation and rebuilding of telomeres, is suppressed in most somatic tissues postnatally. Recent studies have revealed that telomere length is established early in pre-implantation development by a specific genetic programme and correlates with telomerase activity (84). 289 Rev. sci. tech. Off. int. Epiz., 24 (1) Transgenic animals in agriculture Carcass composition Transgenic pigs bearing a human metallothionein promoter/porcine growth-hormone gene construct showed significant improvements in economically important traits such as growth rate, feed conversion and body fat/muscle ratio without the pathological phenotype known from previous growth hormone constructs (69, 78). Similarly, pigs transgenic for the human insulin-like growth factor-I had ~30% larger loin mass, ~10% more carcass lean tissue and ~20% less total carcass fat (79). The commercialisation of these pigs has been postponed due to the current lack of public acceptance of genetically modified foods. Recently, an important step towards the production of more healthful pork has been made by the creation of the first pigs transgenic for a spinach desaturase gene that produces increased amounts of non-saturated fatty acids. These pigs have a higher ratio of unsaturated to saturated fatty acids in striated muscle, which means more healthful meat since a diet rich in non-saturated fatty acids is known to be correlated with a reduced risk of stroke and coronary diseases (66, 83). Lactation The physicochemical properties of milk are mainly due to the ratio of casein variants, making these a prime target for the improvement of milk composition. Transgenic mice have been developed with various modifications demonstrating the feasibility of obtaining significant alterations in milk composition in larger animals, but at the same time, showing that unwanted side effects can occur (50, 89). Dairy production is an attractive field for targeted genetic modification (44, 106). It may be possible to produce milk with a modified lipid composition by modulating the enzymes involved in lipid metabolism, or to increase curd and cheese production by enhancing expression of the casein gene family in the mammary gland. The bovine casein ratio has been altered by over-expression of betaand kappa-casein, clearly underpinning the potential for improvements in the functional properties of bovine milk (8). Furthermore, it may be possible to create ‘hypoallergenic’ milk by knockout or knockdown of the β-lactoglobulin gene; to generate lactose-free milk by a knockout or knockdown of the α-lactalbumin locus, which is the key molecule in milk sugar synthesis; to produce ‘infant milk’ in which human lactoferrin is abundantly available; or to produce milk with a highly improved hygienic standard by increasing the amount of lysozyme. Lactose-reduced or lactose-free milk would render dairy products suitable for consumption by the large numbers of adult humans who do not possess an active intestinal lactase enzyme system. Although lactose is the main osmotically active substance in milk and a lack thereof could interfere with milk secretion, a lactase construct has been tested in the mammary gland of transgenic mice. In hemizygous mice, this reduced lactose contents by 50% to 85% without altering milk secretion (43). Unfortunately, mice with a homozygous knockout for α-lactalbumin could not nurse their offspring because of the high viscosity of their milk (89). These findings demonstrate the feasibility of obtaining significant alterations in milk composition by applying the appropriate strategy. In the pig, transgenic expression of a bovine lactalbumin construct in sow milk has been shown to result in higher lactose contents and greater milk yields, which correlated with improved survival and development of piglets (100). Any increased survival of piglets at weaning would provide significant commercial advantages to the producer. Wool production Transgenic sheep carrying a keratin-IGF-I construct showed that expression in the skin and the amount of clear fleece was about 6.2% greater in transgenic than in nontransgenic animals. No adverse effects on health or reproduction were observed (22, 23). Approaches designed to alter wool production by transgenic modification of the cystein pathway have met with only limited success, although cystein is known to be the ratelimiting biochemical factor for wool growth (96). Environmentally friendly farm animals Phytase transgenic pigs have been developed to address the problem of manure-related environmental pollution. These pigs carry a bacterial phytase gene under the transcriptional control of a salivary-gland-specific promoter, which allows the pigs to digest plant phytate. Without the bacterial enzyme, phytate phosphorus passes undigested into manure and pollutes the environment. With the bacterial enzyme, fecal phosphorus output was reduced by up to 75% (30). Developers expect these environmentally friendly pigs to enter commercial production in Canada within the next few years. Transgenic animals and disease resistance Transgenic strategies to increase disease resistance In most cases, susceptibility to pathogens originates from the interplay of numerous genes; in other words, susceptibility to pathogens is polygenic in nature. Only a 290 few loci are known that confer resistance against a specific disease. Transgenic strategies to enhance disease resistance include the transfer of major histocompatibility-complex genes, T-cell-receptor genes, immunoglobulin genes, genes that affect lymphokines or specific disease-resistance genes (64). A prominent example for a specific disease resistance gene is the murine Mx-gene. Production of the Mx1-protein is induced by interferon and was discovered in inbred mouse strains that are resistant to infection with influenza viruses (88). Microinjection of an interferon- and virus-inducible Mx-construct into porcine zygotes resulted in two transgenic pig lines that expressed the Mx-messenger RNA (mRNA); unfortunately, no Mx protein could be detected (65). Recently the bovine MxI gene was identified and shown to confer antiviral activity as a transgenic construct in Vero cells (6). Transgenic constructs bearing the immunoglobulin-A (IgA) gene have been successfully introduced into pigs, sheep and mice in an attempt to increase resistance against infections (57). The murine IgA gene was expressed in two transgenic pig lines, but only the light chains were detected and the IgA-molecules showed only marginal binding to phosphorylcholine (57). High levels of monoclonal murine antibodies with a high binding affinity for their specific antigen have been produced in transgenic pigs (97). Attempts to increase ovine resistance to Visna virus infection via transgenic production of Visna envelope protein have been reported (15). The transgenic sheep developed normally and expressed the viral gene without pathological side effects. However, the transgene was not expressed in monocytes, the target cells of the viral infection. Antibodies were detected after an artificial infection of the transgenic animals (15). It has also proved possible to induce passive immunity against an economically important porcine disease in a transgenic mouse model (10). The transgenic mice secreted a recombinant antibody in milk that neutralised the corona virus responsible for transmissible gastroenteritis (TGEV) and conferred resistance against TGEV. Strong mammarygland-specific expression was achieved for the entire duration of lactation. Verification of this work in transgenic pigs is anticipated in the near future. Knockout of the prion protein is the only secure way to prevent infection and transmission of spongiform encephalopathies like scrapie or bovine spongiform encephalopathy (98). The first successful targeting of the ovine prion locus has been reported; however, the cloned lambs carrying the knockout locus died shortly after birth (24). Cloned cattle with a knockout for the prion locus have also been generated (19). Transgenic animals with modified prion genes will be an appropriate model for studying the epidemiology of spongiform encephalopathies in humans and are crucial for developing Rev. sci. tech. Off. int. Epiz., 24 (1) strategies to eliminate prion carriers from farm animal populations. Transgenic approaches to increase disease resistance of the mammary gland The levels of the anti-microbial peptides lysozyme and lactoferrin in human milk is many times higher than in bovine milk. Transgenic expression of the human lysozyme gene in mice was associated with a significant reduction of bacteria and reduced the frequency of mammary gland infections (59, 60). Lactoferrin has bactericidal and bacteriostatic effects, in addition to being the main iron source in milk. These properties make an increase in lactoferrin levels in bovine transgenics a practical way to improve milk quality. Human lactoferrin has been expressed in the milk of transgenic mice and cattle at high levels (46, 76) and is associated with an increased resistance against mammary gland diseases (93). Lycostaphin has been shown to confer specific resistance against mastitis caused by Staphylococcus aureus. A recent report indicates that transgenic technology has been used to produce cows that express a lycostaphin gene construct in the mammary gland, thus making them mastitis-resistant (95). Emerging transgenic technologies Lentiviral transfection of oocytes and zygotes Recent research has shown that lentiviruses can overcome previous limitations of viral-mediated gene transfer, which included the silencing of the transgenic locus and low expression levels (104). Injection of lentiviruses into the perivitelline space of porcine zygotes resulted in a very high proportion of piglets that carried and expressed the transgene. Stable transgenic lines have been established by this method (36). The generation of transgenic cattle by lentiviruses requires microinjection into the perivitelline space of oocytes and has a lower efficiency than that obtained in pigs (37). Lentiviral gene transfer in livestock promises unprecedented efficiency of transgenic animal production. Whether the multiple integration of lentiviruses into the genome is associated with unwanted side-effects like oncogene activation or insertional mutagenesis remains to be investigated. Chimera generation via injection of pluripotent cells into blastocysts Embryonic stem cells with pluripotent characteristics have the ability to participate in organ and germ cell development after injection into blastocysts or by aggregation with morulae (81). True ES cells (that is, those 291 Rev. sci. tech. Off. int. Epiz., 24 (1) able to contribute to the germ line) are currently only available from inbred mouse strains (48). In mouse genetics, ES cells have become an important tool for generating gene knockouts, gene knockins and large chromosomal rearrangements (25). Embryonic stem-like cells and primordial germ cell cultures have been reported for several farm animal species, and chimeric animals without germ line contribution have been reported in swine (1, 87, 99) and cattle (13). Recent data indicate that somatic stem cells may have a much greater potency than previously assumed (42, 49). Whether these cells will improve the efficiency of chimera generation or somatic nuclear transfer in farm animals has yet to be shown (48). Culture of spermatogonia and transplantation into recipient males Transplantation of genetically altered primordial germ cells into the testes of host male animals is an alternative approach to generating transgenic animals. Initial experiments in mice showed that the depletion of endogenous spermatogenesis by treatment with busulfan prior to transplantation is effective and compatible with recolonisation by donor cells. Recently, researchers succeeded in transmitting the donor haplotype to the next generation after germ-cell transplantation (38). The major obstacles to this strategy are the lack of efficient in vitro culture methods for primordial germ/prospermatogonial cells, and the limitations this imposes on gene transfer techniques for these cells. Ribonucleic acid interference Ribonucleic acid interference is a conserved posttranscriptional gene regulatory process in most biological systems, including fungi, plants and animals. Common mechanistic elements include siRNAs with 21 to 23 nucleotides, which specifically bind complementary sequences on their target mRNAs and shut down expression. The target mRNAs are degraded by exonucleases and no protein is translated (75). The RNA interference seems to be involved in gene regulation by controlling/suppressing the translation of mRNAs from endogenous viral elements. The relative simplicity of active siRNAs has facilitated the adoption of this mechanism to generate transient or permanent knockdowns for specific genes. For transient gene knockdown, synthetic siRNAs can be transfected into cells or embryonic stages. For stable gene repression, the siRNA sequences must be incorporated into a gene construct (14). The conjunction of siRNA and lentiviral vector technology may soon provide a method with high gene transfer efficiency and highly specific gene knockdown for livestock. Safety aspects and outlook Biological products from any animal source are unique, and must be handled in a different manner from chemically synthesised drugs to assure their safety, purity and potency. Proteins derived from living systems are heat labile, subject to microbial contamination, can be damaged by shear forces and have the potential to be immunogenic and allergenic. In the USA, the FDA has developed guidelines to assure the safe commercial exploitation of recombinant biological products. A crucial consideration with animal-derived products is the prevention of transmission of pathogens from animals to humans (47). This requires sensitive and reliable diagnostic and screening methods for the various types of pathogenic organisms. Furthermore, it should be kept in mind that all transgenic applications of farm animals will require strict standards of ‘genetic security’ and reliable, sensitive methods for molecular characterisation of the products. A major contribution towards the goal of well-defined products will come from matrix-assisted laser desorption/ionisation time-of-flight spectometry (40, 91). Meanwhile improvements in RNA isolation and unbiased global amplification of picogram amounts of mRNA enable researchers to analyse RNA even from single embryos (7). This technology can offer insights into the entire transcriptome of a transgenic organism and thereby ensure the absence of unwanted side-effects (40, 91). Rigorous control is also required to maintain the highest possible levels of animal welfare in cases of genetic modification. Conclusion Throughout history, animal husbandry has made significant contributions to human health and well-being. The convergence of recent advances in reproductive technologies (in vitro production of embryos, sperm sexing, somatic nuclear transfer) with the tools of molecular biology (gene targeting and array analysis of gene expression) adds a new dimension to animal breeding (68). Major prerequisites for success and safety will be the continuous refinement of reproductive biotechnologies and a rapid completion of genomic sequencing projects in livestock. The authors anticipate that within the next five to seven years genetically modified animals will play a significant role in the biomedical arena, in particular via the production of valuable pharmaceutical proteins and the supply of xenografts (Table II). Agricultural applications are already being prepared (39), but general public acceptance may take as long as ten years to achieve. As the complete genomic sequences of all farm animals become available, it will be possible to refine targeted genetic modification in animal breeding and to develop strategies to cope with future challenges in global agricultural production. 292 Glossary Artificial chromosome A construction composed of the basic elements of a normal chromosome into which one or more transgene sequences may be introduced. After introduction into the nucleus, this artificial chromosome is replicated and distributed to daughter cells just like a normal chromosome. The transgene expression is more predictable as it is not influenced by integration into an unknown location. Expression pattern A representation of relative levels of gene expression in the various cells and tissues of a transgenic animal. This is primarily determined by the promoter, but is also dependent on the position in the genome where the construct has become integrated. As cells differentiate, certain chromosomal regions are permanently turned off. Homologous recombination A transgene sequence is constructed which matches the sequence of the endogenous target gene. This biases insertion to the target gene. The design of the gene construct determines whether the target gene will be rendered inactive (knocked out) or simply modified. Inducible promoter A promoter that can be activated by an environmental signal such as the introduction of a heavy metal or an antibiotic. Rev. sci. tech. Off. int. Epiz., 24 (1) percentage of these injected eggs, the transgene becomes incorporated into one of the chromosomes and so will be present in all subsequent cell divisions and in the germ cells, so that future offspring will also carry the transgene. Mosaicism The situation where an individual is composed of cells of more than one genetic background. Transgene integration takes place after the first round of cell division; thus, only a portion of the cells in the resulting individual will be transgenic. It is important that the germ cells carry the transgene so that subsequent offspring will also be transgenic. Multiple transgenic A transgenic organism carrying more than one transgene. This can be achieved either by introduction of several transgenes at the same time, or by obtaining cells or embryos from an existing transgenic animal and using these for a second round of transgenesis. This is most efficiently achieved by nuclear-transfer-based transgenesis because the transfected cells can be screened prior to the production of an embryo. Promoter The control sequence that determines in which tissue or cell type and at which point in time a gene will be expressed. Some promoters are universally active, and some are active only in specific cell types or developmental stages. Reporter gene Knockout Removal of a gene or part of a gene to eliminate a particular protein product. Lentivirus One of several types of virus that have been evaluated for their ability to carry genes into early-stage embryos for the purpose of introducing a transgene. The transgene is placed within the viral genome and is carried into the target embryo by the viral infection mechanism. A sequence that codes for a detectable product such as a fluorescent protein or an enzyme which produces a visible product. Somatic nuclear transfer In its most common form, this involves removal of the nucleus from a fertilised egg and replacement with the nucleus of a differentiated somatic cell such as a fibroblast. The result is a cloned embryo that has the genetic composition of the nuclear donor cell. Spermatogonia Microinjection A now routine procedure in which a glass micropipette is used to deliver a small amount of DNA solution (the transgene) into the nucleus of a fertilised egg. In a small Early developmental precursors of sperm that can be removed from the testes, transfected with a transgene, and then re-implanted to develop into mature sperm that carry the transgene. 293 Rev. sci. tech. Off. int. Epiz., 24 (1) Targeted integration Transfection Sequences have been discovered (FLP, Cre/lox) that cause the insertion or removal of intervening gene sequences. When properly used, these systems can direct insertion to a specific location in the genome, or can at some point in the future remove portions of a transgene that are no longer needed (such as the antibiotic resistance sequences used to select transfected cells). A process mediated by an electrical pulse (electroporation), or by treatment with agents that make the cell membranes porous, such that DNA molecules in the culture medium are able to pass into the cell where they can be expressed or integrated into the genome. Animaux d’élevage transgéniques : le présent et l’avenir H. Niemann, W. Kues & J.W. Carnwath Résumé Jusqu’à une date récente, la microinjection pronucléaire d’acide désoxyribonucléique (ADN) était la méthode standard utilisée pour la production d’animaux transgéniques. Cette technique est actuellement remplacée par des protocoles plus efficaces basés sur le transfert nucléaire de cellules somatiques permettant également de réaliser des modifications génétiques ciblées. De même, les vecteurs lentiviraux et la technique de l’acide ribonucléique (ARN) interférent deviennent des outils importants de la transgenèse. Les animaux d’élevage transgéniques ont une importance en médecine humaine comme sources de protéines biologiquement actives, comme donneurs pour la xénotransplantation et à des fins de recherche en thérapie cellulaire et génique. Parmi les applications agricoles habituelles figurent l’amélioration de la composition des viandes, de la production laitière et lainière, de la résistance aux maladies, de même que la réduction de l’impact sur l’environnement. La sécurité sanitaire des produits peut être assurée par la standardisation des procédures et contrôlée par l’application de la réaction en chaîne par polymérase et de la technologie du criblage de filtres à ADN. Avec l’affinement des données relatives aux séquences et des cartes génomiques des animaux d’élevage, la suppression ou la modification de certains gènes devient de plus en plus réalisable. Cette méthode de reproduction d’animaux jouera un rôle déterminant dans la résolution des problèmes mondiaux qui se poseront demain en matière de production agricole. Mots-clés Animaux d’élevage – Application agricole – Avantage environnemental – « Gene pharming » – Microinjection – Séquence courte à interférence ARN – Transfert nucléaire – Transgénique – Vecteur lentiviral – Xénotransplantation. 294 Rev. sci. tech. Off. int. Epiz., 24 (1) Presente y futuro del ganado transgénico H. Niemann, W. Kues & J.W. Carnwath Resumen Hasta hace poco tiempo, el procedimiento de rigor para obtener animales transgénicos era la microinyección pronuclear de ácido desoxirribonucleico (ADN). Ahora se empiezan a aplicar otros métodos más eficaces basados en la transferencia de núcleos de células somáticas, que además permiten inducir cambios genéticos de manera más específica. El uso de vectores lentivíricos y la técnica del ARN interferente pequeño son otros dos métodos de transgénesis cada vez más empleados. El ganado transgénico es importante en medicina humana porque de él se extraen proteínas biológicamente activas y tejidos u órganos para xenotrasplantes y porque proporciona material para investigar terapias celulares y génicas. En el terreno de la producción animal, sus aplicaciones van desde la mejora de la composición de carne, el rendimiento lechero y la producción de lana hasta el aumento de la resistencia a las enfermedades o la atenuación de los efectos ambientales de la ganadería. La estandarización de protocolos puede servir para garantizar la inocuidad de los productos obtenidos, y técnicas como la reacción en cadena de la polimerasa (PCR) o la de matrices (arrays) de ADN para efectuar los correspondientes controles. A medida que se perfeccionan los mapas genómicos del ganado y se conoce mejor su secuencia génica va resultando cada vez más fácil eliminar o modificar genes concretos. En el futuro, estos sistemas de selección animal serán básicos para resolver los problemas de producción agropecuaria en el plano mundial. Palabras clave Aplicación agropecuaria – ARN interferente pequeño – Beneficio ambiental – Ganado – “Gen pharming” – Microinyección – Transferencia nuclear – Transgénico – Vector lentivírico – Xenotrasplante. References 1. Anderson G.B. (1999). – Embryonic stem cells in agricultural species. In Transgenic animals in agriculture (J.D. Murray, G.B. Anderson, A.M. Oberbauer & M.M. McGloughlin, eds). CABI Publishing, New York, 57-66. 2. Anon. 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Kues,* Reinhard Schwinzer,† Dagmar Wirth,‡ Els Verhoeyen,‡,1 Erika Lemme,* Doris Herrmann,* Brigitte Barg-Kues,* Hansjörg Hauser,‡ Kurt Wonigeit,†,2 and Heiner Niemann*,2 *Department of Biotechnology, Institute for Animal Breeding (FAL), Mariensee, Germany; †Klinik für Viszeral- und Transplantationschirurgie, Medizinische Hochschule Hannover, Hannover, Germany; and ‡Molecular Biotechnology, Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, Germany ABSTRACT The applicability of tightly regulated transgenesis in domesticated animals is severely hampered by the present lack of knowledge of regulatory mechanisms and the long generation intervals. To capitalize on the tightly controlled expression of mammalian genes made possible by using prokaryotic control elements, we have used a single-step transduction to introduce an autoregulative tetracycline-responsive bicistronic expression cassette (NTA) into transgenic pigs. Transgenic pigs carrying one NTA cassette showed a mosaic transgene expression restricted to single muscle fibers. In contrast, crossbred animals carrying two NTA cassettes with different transgenes, revealed a broad tissue-independent and tightly regulated expression of one cassette, but not of the other one. The expression pattern correlated inversely with the methylation status of the NTA transcription start sites indicating epigenetic silencing of one NTA cassette. This first approach on tetracycline regulated transgene expression in farm animals will be valuable for developing precisely controlled expression systems for transgenes in large animals relevant for biomedical and agricultural biotechnology.—Kues, W.A., Schwinzer, R., Wirth, D., Verhoeyen, E., Lemme, E., Hermann, D., Barg-Kues, B., Hauser, H., Wonigeit, K., and Niemann, H. Epigenetic silencing and tissue independent expression of a novel tetracycline inducible system in doubletransgenic pigs. FASEB J. 20, E000 –E000 (2006) Key Words: autoregulative cassette 䡠 bicistronic 䡠 transgenic livestock 䡠 tissue independent expression 䡠 DNA methylation Transgenic mice and farm animals harboring the first generation of conditional promoter elements, which were responsive to heavy metals or steroid hormones, suffered from high basal expression levels and pleiotropic effects (1– 4). In contrast, binary expression systems based on prokaryotic control elements, which are responsive to exogenous IPTG, RU-486, ecdysone, or tetracycline (tet) derivatives are compatible with a 0892-6638/06/0020-0001 © FASEB tightly controlled expression (5, 6). Several transgenic mouse lines expressing modified tet-system components in various tissues have been generated and a rapid and reversible transgene regulation has been shown (7–13). Factors affecting the efficiency of tetracycline-dependent gene regulation include the strain of mice and the site of integration (14), as well as clearance of tetracycline derivatives (15). A high degree of variability in activator-dependent target gene expression is observed and high background expression makes it necessary to screen for appropriately responding lines (16). The original tetracycline system requires two DNA constructs for expression of the transactivator and transactivator dependent expression of the target gene, respectively. These DNA-constructs are usually integrated in two different lines of transgenic mice. On breeding, offspring are obtained in which the target gene can be regulated by addition of doxycycline (7). The long-generation intervals make this approach unfeasible in other mammalian species, including livestock (17). One approach to overcome this limitation is to combine both transactivator and target genes, including individual promoters in a single construct. This approach was compatible with efficient control of gene expression in mammalian cells and mice (18, 19). However, the near physical distance of two promoter sequences makes this system prone to elevated background expression. A tetracycline-controlled transcriptional silencer has been used to suppress background expression (9). Alternatively, autoregulatory expression 1 Present address: Envelope retrovirale et ingeniérerie retrovirale, INSERM 758, ENS de Lyon, France. 2 Correspondence: K. W., Klinik f. Viszeral-und Transplantations Clinique, Medizinische Hochschule Hannover, CarlNeuberg Str. 1, Hannover D-30623, Germany. E-mail: [email protected] H. N., Institut for Animal Breeding (FAL) Department of Biotechnology Höltystr. 10, Neustadt D-31535, Germany. E-mail: [email protected] doi: 10.1096/fj.05-5415fje E1 systems have been established that overcome the problem of promoter interference. In these systems the transactivator is either expressed from a tet-responsive bicistronic expression cassette (20 –23) or from a tetresponsive bidirectional promoter (24, 25). It has been shown that expression of biologically active proteins can be tightly controlled with these systems, albeit minimum basal expression levels of the transactivator are required (26). Another advantage of autoregulative cassettes is that transgene transcription is linked to ubiquitously present cellular cofactors, thereby circumventing cell-/tissue-specific expression as observed for classical promoters. Here, we describe tightly controlled transgene overexpression in the domestic pig by an autoregulative bicistronic tetracycline-responsive cassette (NTA). The NTA-cassette was designed to ensure high and ubiquitous expression levels of human regulators of complement activation (RCA) relevant for xenotransplantation (17), (i.e., decay accelerating factor (DAF)) and CD59. The rationale for this approach was twofold: (i) As proof of principle we wanted to demonstrate that single-construct cassettes can be successfully expressed in large animals; (ii) we intended to improve xenotransplantation by producing tet-off regulated human complement regulator transgenic animals. Unexpectedly, nine independent transgenic pig lines showed expression restricted to muscle fibers. However, in crossbred double-transgenic animals a broad tissue independent transgene expression of the CD59-NTA cassette, but not of the DAF-NTA cassette was discovered. This asymmetric expression pattern correlated with different methylation patterns of the NTA cassettes indicating that epigenetic mechanisms are critical for the function of the NTA system. Expression of human complement regulators and tet transactivator did not compromise animal health and fertility. provided by A. Saalmüller, Wien, Austria) was carried out using essentially the same protocol as for NIH3T3 cells. Flow cytometry was performed as described earlier (27) using specific monoclonal antibodies (PharMingen, Hamburg, Germany) to detect CD59 and DAF. MATERIALS AND METHODS Detection of CD59 and DAF expression Construction of the bicistronic NTA-cassettes Transgenic offspring and wild-type control animals were sacrificed and tissue samples were obtained from the following organs: heart, lung, liver, kidney, spleen, thymus, skeletal muscle, pancreas, skin, brain, and aorta. Samples were used for primary cell cultures or snap-frozen in liquid nitrogen and used for RT–PCR, Northern blotting and immunohistological analysis. For RT–PCR, total RNA was isolated using the guanidinium thiocyanate-phenol-chloroform extraction. PCR detection and Northern blotting of CD59 and DAF transcripts were done as described recently (30). In brief, digoxigenin labeled 605 and 613 bp antisense cRNAs for CD59 and DAF were used for specific detection of the transgenic transcripts. The calculated sizes for the bicistronic mRNAs were 2.3 and 3.0 kb for the CD59 and DAF encoding transcripts, respectively. In addition to the 3.0 kb band a slightly faster migrating fragment was specifically detected in the DAF Northern. Apparently, the smaller fragment represented a degradation product of the 3.0 kb mRNA. In some experiments tissues collected from transgenic animals carrying a cytomegalovirus (CMV) promoter-CD59 construct were used as controls (30). A -actin (Actb) probe (31) served as an internal control. CD59 was amplified from pWTCD59 (27). DAF was amplified from pWTDAF (27). The transactivator tTA was amplified from pUHD15–1 (28), thereby flanking the tTA cDNA with NotI and PstI sites. The cDNAs were independently integrated into pTBC-1 (29) and both plasmids were then fused via the unique BamHI and VspI sites. All polymerase chain reaction (PCR)-derived sequences were verified by DNA sequencing. Vector sequences are available on request. Cell culture and transfection NIH3T3 cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM). The RCA-NTA cassettes were cotransfected with a pAG60 by DNA-calcium phosphate coprecipitation (27). Cells were selected for resistance to G418 (1 mg/ml). Pools and cell clones were generated and cultivated in the presence and absence of doxycycline (2 g/ml). Transfection of swine testis epitheloid (STE) cell line (kindly E2 Vol. 20 June 2006 Determination of doxycyline plasma levels in pigs A mouse-derived cell line NIH-TAluc with a stably integrated doxycycline-responsive luciferase cassette was used to determine doxycycline levels in plasma and fodder extracts. Generation of transgenic pigs Transgenic pigs were generated by pronuclear DNA microinjection as described (30). In brief, zygotes were collected from hormonally pretreated prepubertal German landrace donor gilts 15–18 h after the second mating. Animals were sacrificed, and zygotes were flushed from the excised oviducts. The NTA constructs were released from vector backbones by digesting the plasmids with BsrBI and Tth111I (New England Biolabs (NEB), Frankfurt, Germany) and isolated by gel electrophoresis. Approximately 10 pl of purified DNAs (4 –10 ng/l) were injected into one pronucleus of zygotes after centrifugation for 6 min at 14,500 g to visualize the pronuclei by concentrating the cytoplasmic lipids at one pole. A total of 653 microinjected zygotes were surgically transferred into the oviducts of 20 synchronized prepubertal gilts, resulting in 80 piglets from which 11 could be identified as transgenic by Southern blotting of the genomic DNA. For Southern blotting, the genomic DNA was digested with PstI (NEB) and blotted to Hybond membranes. The PstI digest released internal fragments from the NTA-cassettes, which were 2049 and 1658 bp for the CD59 and DAF-constructs, respectively. The complete constructs were isolated from vector backbones and used as probes after digoxigenin labeling. Hybridization products were visualized by chemiluminescence. Founder animals were bred with nontransgenic animals to test for germline transmission. Transgenic F1 and F2-animals were used for detailed analysis of the expression pattern and doxycycline responsiveness. Animals were treated according to institutional guidelines and experiments were approved by an external animal welfare committee. The FASEB Journal KUES ET AL. To detect CD59 and DAF, immunohistological staining was performed on cryostat sections using biotinylated anti-human mAB H19 (CD59) and mAB33572X (DAF/CD55), respectively (both antibodies were from BD PharMingen). Human heart tissue served as positive control. Specific antibody (Ab) binding was detected by incubation with streptavidine-peroxidase and 3-amino-9-ethylcarbazole (AEC) as substrate, cell nuclei were counterstained with hematoxylin. Tissue samples were evaluated at 100 –200⫻ magnifications. Primary cell cultures of porcine fibroblasts were established from subdermal tissues as described (30, 32) and employed for expression analysis by Northern blotting or flow cytometry (FACS). Genomic DNA was isolated from porcine tissues by proteinase K lysis. The following primers spanning the complete promoter regions of DAF-NTA and CD59-NTA constructs were used to amplify these regions by PCR: CMV-0, 5⬘-AAA ATA GGC GTA CAC GAG G; hDAF-1, 5⬘-ATC ACT GAG TCC TTC TCG CC and hCD59 –1, 5⬘-CCC TCA AGC GGG TTG TGA CG. The PCR products were directly sequenced, employing the CMV-O primer. ers were used to amplify the CMV minimal promoter of the CD59-NTA construct, CMV-2bi, 5⬘- GAA AGT TGA GTT CGG TAT TT, CD59 –2bi, 5⬘- AAA AAT ATC CCA CCT TTT TC; for the DAF-NTA construct the CMV-2bi primer in combination with DAF-2bi, 5⬘- CCA TTA ACT ACC CTT AAA AC was used. RESULTS Evaluation of autoregulatory RCA–NTA expression cassettes in cell lines Doxycycline was fed in the form of tablets (doxy 200®, CT Arzneimittel GmbH, Berlin, Germany; one tablet contains 200 mg doxycycline). A daily dose of 3.3 mg/kg body weight was applied for periods from 6 to 45 d. In one feeding group, blood samples were taken during the course of the experiment to determine doxycycline levels in plasma. Muscle biopsies from the M. longissimus were obtained by a special biopsy device routinely used in meat quality testing program (33). Biopsies were taken from the longissimus muscle prior to doxycycline treatment, and at different time points thereafter and were analyzed by Northern blotting and immunohistology. In some experiments, subdermal tissue pieces from the biopsies were used to establish primary cell cultures. We constructed an autoregulatory bicistronic tet-off based cassette that supports single step transduction (Fig. 1A) and in which the mRNA is driven by the tet responsive promoter (PtTA). In two constructs carrying either the human CD59 or DAF cDNAs (designated CD59-NTA and DAF-NTA) the RCA reading frames are followed by an internal ribosomal entry site (IRES) that provides translation of the recombinant transactivator (tTA). Except for the human cDNAs, both cassettes were completely identical. Mouse NIH 3T3 cells stably expressing the cassettes showed autoactivation and expression of the respective RCA (Fig. 1B). Expression was efficiently suppressed by addition of doxycycline to the culture medium (Fig. 1B). In addition, swine testis epitheloid (STE) cells were transfected with the RCA-NTA cassettes, and again autoactivation and conditional transgene regulation were found (not shown). Thus, autoregulatory bicistronic expression cassettes support tight regulation of human RCAs in xenogenic cells, suggesting that ubiquitous activation could be achieved in vivo. Bioinformatic analysis of CpG islands Generation of RCA-NTA transgenic pigs For CpG island prediction the MethPrimer program (http:// www.urogene.org/methprimer) was employed. The NTA-cassette sequences were analyzed using the following criteria for CpG island prediction: i) a CG content greater than 60%, ii) a presence of CpG dinucleotides of greater than 0.6, and iii) a DNA region of greater than 300 bp. Five hCD59-NTA and six DAF-NTA transgenic founder animals were generated by pronuclear microinjection of the respective cassette into porcine zygotes, from which ten transgenic lines were established. Transgenic juvenile and adult F1 and F2-offspring were used for expression profiling in the absence of any tetracycline. Remarkably, an intensive immunostaining indicative for human RCA protein was found in single fibers of striated muscle in 9 of the 10 lines (Table 1, Fig. 1C; 2–3 animals investigated per line). Depending on the line, 5–30% of the muscle fibers stained positive. Expression in muscle was confirmed by Northern blotting with DAF and CD59 specific probes, respectively. Contrary to the tissue-independent design of the NTA cassette, Northern blotting, immunohistology, and FACS revealed that the animals did not express human RCAs in brain, heart, kidney, tongue, liver, skin, lymph nodes, blood and pancreas (summarized in Table 1). Apparently, the (different) integration sites of the nine expressing lines had no or only a marginal effect on the muscle fiberspecific expression. Integration of the entire bicistronic cassettes into the porcine genome was confirmed by Southern blotting of genomic DNA digested with restriction enzymes, cut- Application of doxycycline Detection of methylated CpG dinucleotides by bisulfite sequencing Bisulfite sequencing was done as described by Hajkova et al. (34). In brief, 20 ng genomic DNA was digested with EcoRI (NEB). Denatured DNA was embedded in 7 l of 20 mg/ml low melting agarose (Biozym, Hess. Oldendorf, Germany), cooled to form agarose beads, and incubated in 2.5 M bisulfite-hydroquinone solution pH 5 (Roth, Hamburg, Germany) for 4 h at 50°C. PCR was carried out in a final vol of 100 l with individual agarose beads containing embedded genomic DNA. PCR conditions were as follows: 1 ⫻ PCR reaction buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl2), 1.5 mM MgCl2, 0.2 M dNTPs (Amersham Biosciences Europe, Freiburg, Germany), and 0.6 M of each primer for 40 cycles. The PCR fragments were separated by gel electrophoresis, isolated and directly sequenced. Only sequences with ⬎ 95% cytosine conversion of non-CpGs were analyzed. Methylation status of CpG dinucleotides was determined based on the ratio of cytosines to converted cytosines. The following prim- INDUCIBLE TRANSGENIC EXPRESSION IN A LARGE ANIMAL MODEL E3 Figure 1. Design of the autoregulative NTA expression cassette and conditional expression of transgenes in cell lines and in singletransgenic pigs. A) The PtTA promoter (28) drives a bicistronic mRNA encoding a human regulator of complement activation (RCA) linked with a tet-off transactivator (tTA) via a poliovirus IRES. Two constructs carrying the human cDNAs encoding either CD59 (CD59NTA) or DAF (DAF-NTA) were used. Minimal expression of the transactivator results in tTA binding to the tet-operator sequences and initiates an autoregulative loop. In the presence of exogenous tetracycline the transactivator is inactivated and transcription silenced. B) Conditional expression of hDAF in cell culture. NIH3T3 cells were stably transfected with DAF-NTA. A cell pool and a single clone (clone 12) were cultivated in presence (green line) or absence (black line) of doxycycline. Cells were harvested and DAF expression was determined by indirect immunofluorescence using flow cytometry. An overlay of the histograms is presented with nontransfected NIH3T3 cells (red line) as a control. C) Muscle fiber confined expression of DAF and CD59 in transgenic pigs bearing a single NTA-cassette. Longitudinal (upper row) and cross sections (lower row) of transgenic and wild-type pig muscles after specific Ab staining are shown, nuclei are counterstained with hematoxylin. Note that only individual fibers displayed intense Ab staining, whereas neighboring fibers are negative. No expression of the transgenes was detected in several other tissues (for details see Table 1). ting the NTA cassette internally near the ends (not shown). To rule out the possibility, that transgenic animals carried defective promoter sequences, the respective elements were amplified from porcine genomic DNA by PCR. Sequencing confirmed that heptamerized tet-operator and CMV basal promoter sequences were present in both, CD59-NTA and DAFNTA cassettes. To further rule out putative contamination of the fodder with tetracycline and/or its derivatives, fodder and plasma samples of transgenic pigs were analyzed via a doxycycline sensitive reporter cell line. All samples proved to be negative for tetracycline activity (data not shown). Enhanced tissue-independent expression of CD59 in crossbred animals carrying two NTA-RCA cassettes To investigate whether the unexpected mosaic expression pattern in striated muscle was due to suboptimal transactivator expression levels, tTA was expressed in porcine primary fibroblasts from transgenic animals. Although cotransfected tTA-responsive reporter genes were activated, the chromosomal RCA-NTA constructs remained unresponsive (data not shown) indicating stable silencing of the chromosomal cassettes. To test if the typical mosaic expression pattern in transgenic pigs could be altered by increased threshold levels of tTA in vivo, heterozygous transgenic animals E4 Vol. 20 June 2006 carrying either CD59-NTA or DAF-NTA were crossbred to obtain double-transgenic animals. From three litters with a total of 24 piglets the expected mendelian distribution of transgene combinations was obtained; i.e., 5 piglets (21%) were double-transgenic (CD59-NTA ⫹ DAF-NTA), 12 (50%) carried either the CD59 or DAF cassette and 7 (29%) were nontransgenic. In addition, a female and male double-transgenic sibling (CD59NTA ⫹ DAF-NTA) were mated and produced nine piglets, i.e., four piglets carrying both cassettes, four piglets had one cassette and one nontransgenic offspring was obtained. From the nine double-transgenic animals, three were sacrificed at the age of 6 wk and 6 mo, respectively, and organs were analyzed for expression of the transgenes. Muscle biopsies and blood samples were analyzed from the other double-transgenic animals. Interestingly, a selective up-regulation of CD59 expression was found in double-transgenic animals derived from crossbreeding L13 and L20 (Table 1). The CD59 mRNA levels were significantly (50 –100 fold) elevated in three different muscles, i.e., Musculus (M.) longissimus, front and hind leg muscles, in double-transgenic animals over transgenic siblings carrying only one CD59-NTA cassette (Fig. 2A). Immunohistological staining revealed that virtually all fibers in striated muscle stained positive for CD59 (Fig. 3A). In addition, other organs, including liver, pancreas, kidney and lung expressed the transgene at the mRNA The FASEB Journal KUES ET AL. TABLE 1. Expression patterns in animals carrying one and two NTA cassettes Transgenic animals with two cassettes DAF-NTA ⫻ CD59-NTA Transgenic lines bearing one NTA cassette DAF-NTA Heart Liver Lung Kidney Pancreas Skeletal mus. Thymus Lymphocytes Fibroblasts L13 ⫻ L20 (n⫽7) CD59-NTA L15 ⫻ L20 (n⫽2) L12 L13 L14 L15 L16 L17 L19 L20 L21 L22 DAF CD59 DAF CD59 n.d ⫹⫹ - ⫹ n.d. n.d. ⫹ n.d. n.d. ⫹ n.d. n.d. ⫹ n.d. n.d. ⫹ n.d. n.d. ⫹⫹ - ⫹ n.d. n.d. ⫹ n.d n.d. ⫹⫹ n.d. ⫹* - ⫹ ⫹⫹ ⫹ ⫹ ⫹⫹ ⫹⫹⫹⫹ n.d. ⫹⫹* ⫹ ⫹ n.d. n.d. ⫹⫹ n.d. n.d. Summarized expression data obtained by Northern blotting, immunohistology and FACS; plus signs indicate low (⫹) to high (⫹⫹⫹⫹) levels of expression, –: below detection limit, n.d.: not determined; *after mitogen stimulation. (Fig. 2B) and protein levels (Fig. 3A). RNA and protein expression pattern of CD59 correlated well, e.g., muscle and liver, which showed the highest mRNA levels (Fig. 2A) also displayed the highest proportion of anti-CD59 Ab stained cells (Fig. 3A). Tissues with lower mRNAs levels, such as lung, showed only few positive cells in the immunohistology. Importantly, in all examined tissues the positive cells were strongly stained for CD59, suggesting that the autoregulative up-regulation worked well in this population of cells. However, DAF mRNA and protein levels were not increased in the same double-transgenic animals and immunoreactivity remained confined to single muscle fibers (Fig. 3A). Seven out of nine double-transgenic pigs, derived from crossing transgenic lines L13 with L20 showed this asymmetric over-expression of CD59. The remaining two double-transgenic pigs, derived from crossing L15 with L20, mimicked expression patterns of their parental lines showing mosaic expression in single muscle fibers. Individual muscle fibers coexpressed both CD59 and DAF (Fig. 3B), while some fibers expressed only CD59 (Fig. 3B, arrows), but not DAF (Fig. 3B, circles), highlighting that in these fibers high transactivator levels alone were not sufficient to activate the DAF-NTA gene. Fibers with exclusive expression of DAF were never detected. Since none of the single-transgenic animals showed this enhanced transgene expression, we argue that the presence of two cassettes is necessary, but not always sufficient for tissue independent expression. This finding was confirmed by the widespread expression of CD59 in a CD59-NTA homozygous animal (CD59-NTA ⫹CD59-NTA) (data not shown). Health status, development, and fertility of animals bearing either one or two NTA-cassettes were not compromised. All animals were not distinguishable from their nontransgenic counterparts. Figure 2. Asymmetric over-expression in pigs carrying two cassettes. A) Overexpression of CD59-NTA transcripts in double-transgenic siblings. Northern blot of total RNAs isolated from fore limb (lane 1), hind-limb (lane 2) and M. longissimus (lane 3) of a double-transgenic animal. Lanes 4 – 6 were loaded with RNA from fore-limb, hind-limb, and M. longissimus from a CD59-NTA single-transgenic sibling. Lanes 7 and 8 were loaded with samples from a wild-type animal and lanes 9 and 10 with RNA isolated from a CMV-CD59 transgenic animal described previously (30). The CD59 transcripts in lanes 1–3 are connected via an IRES element with the tet transactivator resulting in a 2.3 kb transcript, whereas the conventional CMV-hCD59 construct (lanes 9, 10) produces a 1.6 kb transcript. B) Tissue specific expression of CD59 and DAF in double-transgenic animals. The gel was loaded with (1) a RNA size marker and total RNA isolated from heart (2), lung (3), liver (4), kidney (5) and skeletal muscle (6) and probed with CD59 and DAF specific probes. INDUCIBLE TRANSGENIC EXPRESSION IN A LARGE ANIMAL MODEL E5 eight weeks after termination of the doxycycline application (Fig. 4). Tightly controlled expression of human RCA in white blood cells To analyze the regulatory capacity of the NTA cassettes, we studied the expression patterns of DAF and CD59 by flow cytometry on white blood cells collected from seven double-transgenic animals (L13⫻L20), and 2 single-transgenic (1 DAF, 1 CD59) pigs. No significant expression of DAF or CD59 was found in lymphocytes (resting and concanavalin A (Con A)-activated cells) from the two single-transgenic pigs. (Fig. 5A). Absence of DAF and CD59 was also typical for resting cells from double-transgenic animals (Fig. 5B), although in one animal a small number of CD59 expressing cells were detected (data not shown). However, expression of DAF and CD59 could be induced in cells from all double-transgenic animals by activation with ConA. The intensity of ConA-mediated up-regulation of the two molecules was asymmetric showing strong expression of CD59 and weak but significant up-regulation of DAF. Expression of both molecules could be terminated by culturing the cells with tetracycline for 48 h (Fig. 5B), again providing evidence for a tight control of gene expression from these cassettes. Identification of CpG islands and determination of the methylation status Figure 3. Asymmetric expression of CD59 and DAF in doubletransgenic animals. A) Asymmetric over expression of CD59 in different organs of double-transgenic animal #335. This animal was produced by breeding L13 with L20. Note the widespread expression of CD59 in muscle, liver and pancreas as indicated by a red-brown precipitate. In contrast, DAF expression is still confined to single muscle fibers (arrows). B) Coexpression of CD59 and DAF in single muscle fibers of double-transgenic animal #364 detected by immunostaining of serial cross sections. This animal was generated by breeding L15 with L20. Note that some fibers expressed exclusively CD59 (arrows), but not DAF (dashed circles). Epigenetic mechanisms were hypothesized to be causatively involved in the asymmetric expression of the two RCA-NTA cassettes in double-transgenic animals and the muscle fiber-specific expression in single-transgenic Conditional RCA-NTA regulation in single- and double-transgenic animals To evaluate the exogenous control of the autoregulated tet-off cassettes, 12 single-transgenic and four double-transgenic animals were fed with doxycycline and muscle biopsies were taken before, during and after antibiotic treatment. A daily dose of 3.3 mg doxycycline/kg body weight for 6 d was found to be effective to shut down transgene expression (Fig. 4). This dose is well below the recommended effective dose of 12 mg doxycycline/kg/day for antibiotic treatment of pigs. RCA-NTA transcripts were virtually eliminated by doxycycline feeding within 2– 6 d. Thus, the expression cassettes located at different chromosomal sites could be readily switched off. Re-expression of RCANTAs took unexpectedly long, and did not resume until E6 Vol. 20 June 2006 Figure 4. Conditional transgene expression by doxycycline feeding. Two double-transgenic pigs (#368, #369) were fed with a daily dose of 3.3 mg/kg doxycycline for 6 d. As control, a third double-transgenic animal (#366) was left untreated. Muscle biopsies were taken at several time points before (day 0), during (day 2), and after (days 13, 19, 75, and 180) the feeding regime, 5 g total RNA was loaded per lane and analyzed by Northern blotting with RCA and -actin (ACTB) specific probes. Note that the RCA-NTA transcripts disappeared already after 2 d of doxycycline feeding. Repression of the RCAs resumed 8 wk after the end of doxycycline feeding. The calculated size of the DAF-IRES-tTA mRNA was 3.0 kb. The FASEB Journal KUES ET AL. pigs. Bioinformatic analysis of the DAF-NTA cassette revealed that the minimal CMV promoter within PtTA and the 5⬘coding region of the DAF cDNA together contained a putative CpG island (Fig. 6A). In contrast, no CpG island was predicted for the PtTA and the 5⬘coding region of the CD59 gene (Fig. 6A). Genomic DNA from muscle biopsies of single and double-transgenic pigs was isolated, and analyzed by bisulfite sequencing to assess the methylation status of the NTA-promoter regions in the pig genome. The promoter region of the CD59-NTA cassette was completely methylated in single-transgenic animals, while in double-transgenic animals only ⬃50% of the CpG dinucleotides were fully methylated (Fig. 6B). In contrast, this region was essentially nonmethylated in CD59-NTA cassettes from stably transfected STE cells. An almost complete methylation of the CpG dinucleotides around the transcription initiation site of the DAF-NTA cassette was discovered either in single or double-transgenic animals (Fig. 6C). Primary fibroblasts isolated from biopsies also maintained this methylation pattern (Fig. 6C). For comparison, the methylation status of the DAF-NTA cassette was determined in a pool of stably transfected NIH 3T3 cells and in two cell clones, which expressed high levels of the DAF-NTA construct. These cells possessed a relative low concentration of methylation (Fig. 6C). Similarly, stably transfected porcine STE cells showed weak methylation of the promoter regions (Fig. 6C). Thus an inverse correlation between the observed gene expression and the methylation status of these cassettes is obvious. These data indicate that the PtTA is prone to methylation in transgenic pigs, in particular in combination with the 5⬘coding region of the human DAF gene. DISCUSSION Here, we report the first conditional and broad transgene expression in a domestic animal species using an autoregulative tet-off system. The methylation of transgene promoters correlated well with expression levels, suggesting de novo methylation as a crucial mechanism for expression of RCA-NTA constructs in these pigs. The bicistronic, autoregulative cassettes were designed Figure 5. Expression patterns of hDAF and hCD59 on lymphocytes from transgenic pigs. The cells were stained by indirect immunofluorescence and analyzed on a FACSCalibur flow cytometer. Closed histograms (thin lines) represent fluorescence intensity obtained after incubation of the cells with mouse anti-human DAF (IA10, IgG2a) or CD59 (H19, IgG2a) monoclonal antibody followed by a second incubation step with FITC-conjugated goat antimouse immunoglobulin (FITC-immunoglobulin). Open histograms (bold lines) were obtained after staining of the cells with FITC-immunoglobulin only. A) Analysis of lymphocytes obtained from a single hCD59 transgenic pig. Resting cells and cells, which had been activated by culturing with Con A plus interleukin (IL)-2 for 48 h were studied. Similar results (absence of hDAF and hCD59) were obtained when cells from a single DAF transgenic pig or cells from wild-type nontransgenic control animals were analyzed. B) Analysis of lymphocytes from a double- transgenic pig. Expression of DAF and CD59 was first examined on resting cells. The cells were then activated for 48 h with Con A plus IL-2 and expression of the two molecules was reanalyzed (activated). Note, the expression of both CD59 and DAF (arrows) in ConA activated cells. Cultivation was extended for additional 48h in the absence or presence of tetracycline (Tet, 1 g/ml). The histograms shown in the bottom panels (⫹Tet) indicated termination of human RCA expression in tetracycline treated cells. Cells which had been cultured for 96 h in the absence of tetracycline showed the same asymmetric expression pattern (weak DAF, strong CD59) as the 48 h activated cell population. INDUCIBLE TRANSGENIC EXPRESSION IN A LARGE ANIMAL MODEL E7 Figure 6. CpG rich regions in NTA promoter regions and methylation status. A) CpG island analysis of the DAF- and CD59-NTA cassettes with the MethPrimer program. The plots depict tet operator region, CMV basal promoter and the RCA coding regions; CpG dinucleotides are indicated by red bars below the plots; putative CpG islands are indicated as solid blue areas. Note that the DAF-NTA construct carries a putative CpG island, which covers the transcription start and the 5⬘coding region. B) Methylation status of the CD59-NTA transcription start region in transgenic pigs and stably transfected STE cells. CpG dinucleotides are depicted as circles: black, fully methylated; gray, half-methylated; white, nonmethylated. C) Methylation status of the DAF-NTA transcription start region in transgenic pigs, primary fibroblasts and stably transfected NIH 3T3 and STE cells. to be compatible with stable and widespread expression and carried either CD59 or DAF, followed by an IRES and the tTA coding sequence. The target genes were chosen, since human CD59 and DAF are critically involved in the suppression of the hyperacute rejection of xenotransplanted porcine organs (35). For autoactivation, a minimum basal expression of the minimal promoter is required. The original axiome of the tet system predicts that in the absence of tetracycline, tTA binds to the tet operator sequences and activates transcription (5, 6, 36). The presumed autoregulated function of the NTA cassettes was found to be fulfilled in NIH3T3 and porcine STE cells. E8 Vol. 20 June 2006 Unexpectedly, transgenic pigs carrying one NTA cassette displayed confined expression in single striated muscle fibers, irrespective of the transgene or the integration site. Mosaic expression patterns have also been described in tet-transgenic mice and, in some cases, the highest expression levels were found in skeletal muscle (7, 16, 18). In the present study, confinement to the muscle compartment could be overcome by crossbreeding selected double-transgenic pigs carrying both the DAF-NTA and CD59-NTA cassettes. This resulted in a widespread expression of CD59 in muscle, liver, pancreas, lung and other tissues, whereas DAF expression remained restricted to individual muscle fibers. The lack of a widespread expression of DAF/NTA correlated with the methylation of critical parts of the tTA dependent promoter and neighboring sequences and is thus epigenetic in nature. Methylation is usually associated with additional changes in histone conformation and chromatin structure, rendering the DNA region into a compressed state and leading to gene silencing (37–39). Although the CD59-NTA transgene did not possess a predicted CpG island, bisulfite sequencing revealed that the CpGs around the transcription start site were fully methylated in single-transgenic animals and that approximately half of the CpG dinucleotides were methylated in double-transgenic animals. Studies with Epstein-Barr virus have shown that even methylation of few sites is sufficient to suppress transcription of a promoter, and experiments with episomes have demonstrated that methylation of as few as 7% of the CpG sites can induce quiescence of a gene locus (40, 41). Why individual skeletal muscle fibers showed a relaxed transgene expression control of the NTA cassettes in single-transgenic animals remains to be investigated. We cannot rule out that simple gene dose effects led to the widespread expression in double-transgenic animals. However, we assume that unknown molecular mechanisms during critical time windows have resulted in the observed widespread expression in double-transgenic pigs. It is well known that genomic methylation and histone modifications undergo major changes during development (42). Our data indicate that de novo methylation may interfere with proper expression of tetracycline sensitive promoters with significant implications for the generation of tet-transgenic animals as most of the employed promoter sequences are based on the CMV minimal promoter. Investigations of DMNT1 over-expressing cell lines favor the concept that the sequence context might play a crucial role in the methylation sensitivity of CpG islands (43). This might explain the high degree of variability in activator-dependent target gene expression requiring screening for appropriately responding mouse lines, as well as the here observed asymmetric expression in pigs. Along this line it is interesting that CpG depleted CMV promoter constructs gave rise to long term expression in vivo (44). Effective conditional transgene expression could be The FASEB Journal KUES ET AL. demonstrated in transgenic pigs and in white blood cells. Feeding of transgenic pigs with a daily dose of 3.3 mg doxycycline per kg body weight terminated transgene transcription in vivo. Re-expression of human RCAs after termination of doxycycline application was resumed by autoactivation after ⬃8 wk. Whether this delayed re-expression is due to a sluggish clearance of doxycycline (15) or the architecture of the autoregulative expression cassette remains to be determined. At present we do not have evidence for specific metabolic features or a tetracycline reservoir in the pig that could explain the long interval from termination to re-expression of the cassette. Measurements of doxycyline levels in porcine plasma indicated a rapid decline after the application. Analysis of the fodder for doxycycline contaminations did not reveal any doxycycline residues. Overall, these data show that the regulation of the RCA-NTA cassettes is tight, once the expression is turned off by doxycycline. Probably, time consuming stochastic events re-express RCA-NTA cassettes and induce autoactivation. However, in cell culture RCAs were re-expressed within 6 –10 d after withdrawal of doxycycline. In conclusion, we have generated transgenic pigs bearing a bicistronic tetracycline-responsive cassette and demonstrated tightly controlled expression in a large animal model for the first time. The usage of the autoregulative bicistronic cassette supports efficient single step transduction, which is of utmost importance in view of the long generation intervals in large domestic animals. Expression of RCAs and transactivator did not compromise health status and fertility of transgenic pigs. Design of the next generation of expression cassettes will take into account potential methylation prone sequences and should thus be compatible with true ubiquitous transgene expression already in the F0-generation. The use of optimized autoregulative constructs in combination with improved gene transfer techniques, such as somatic nuclear transfer or lentiviral vectors will pave the way toward precisely controlled transgene expression in farm animals for biomedical and agricultural application (45). The critical discussions and comments by J.W. Carnwath are gratefully acknowledged. The authors thank K-G. Hadeler, H.H. Döpke (Mariensee) and L. Schöberlein (Leipzig) for expert help with the muscle biopsy device and A. Saalmüller (Wien) for gift of the STE cell line. The excellent technical assistance of A. Kanwischer (Hannover) and W. Mysegades, the introduction into bisulfite sequencing by C. Gebert, as well as the expert animal caretaking by H. Hornbostel and T. Peker are kindly acknowledged. This work was supported by grants of the DFG (SFB 265, Forschergruppe 535) and BMBF to H.N. Support of the Kuratorium für Dialyze und Nierentransplantation to K.W. is acknowledged. Competing financial interests: The authors declare that they have no competing financial interests in relation to this paper. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. REFERENCES 1. Mayo, K.E., Warren, R., and Palmiter, R. D. (1982) The mouse metallothionein-I gene is transcriptionally regulated by cad- 21. INDUCIBLE TRANSGENIC EXPRESSION IN A LARGE ANIMAL MODEL mium following transfection into human or mouse cells. 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Trends Biotechnol. 22, 286 –294 The FASEB Journal Received for publication November 15, 2005. Accepted for publication February 14, 2006. KUES ET AL. Copyright Blackwell Munksgaard 2006 Xenotransplantation 2006: 13: 345–356 Printed in Singapore. All rights reserved doi: 10.1111/j.1399-3089.2006.00317.x XENOTRANSPLANTATION Health status of transgenic pigs expressing the human complement regulatory protein CD59 Deppenmeier S, Bock O, Mengel M, Niemann H, Kues W, Lemme E, Wirth D, Wonigeit K, Kreipe H. Health status of transgenic pigs expressing the human complement regulatory protein CD59. Xenotransplantation 2006; 13: 345–356. Blackwell Munksgaard, 2006 Abstract: Background: Microinjection of foreign DNA into pronuclei of zygotes has been the method of choice for the production of transgenic domestic animals. Following microinjection the transgene is randomly integrated into the host genome which can be associated with insertional mutagenesis and unwanted pathological side effects. Methods: Here, we evaluated the health status of pigs transgenic for the human regulator of complement activation (RCA) CD59 and conducted a complete pathomorphological examination on 19 RCA transgenic pigs at 1 to 32 months of age from nine transgenic lines. Nine wild-type animals served as controls. Expression levels of human complement regulator CD59 (hCD59) mRNA were measured by RT-PCR and distribution of hCD59 protein was determined by immunohistochemistry. Results: Albeit variable transgene expression levels, no specific pathomorphologic phenotype associated with the presence of the transgene in all analyzed pig lines could be detected. Conclusions: Transgenic expression of this human RCA gene construct is not correlated with a specific pathological phenotype in pigs. This is crucial for the application of the technology and the use of transgenic pigs for biomedical and agricultural applications. Stefanie Deppenmeier,1,2 Oliver Bock,2 Michael Mengel,2 Heiner Niemann,3 Wilfried Kues,3 Erika Lemme,3 Dagmar Wirth,4 Kurt Wonigeit5 and Hans Kreipe2 1 Department of Pathology, School of Veterinary Medicine Hannover, Hannover, 2Department of Pathology, Hannover Medical School, Hannover, 3 Department of Biotechnology, Institute of Animal Breeding, Mariensee (FAL), Neustadt, 4Department of Gene Regulation and Differentiation, German Research Center for Biotechnology, Braunschweig, 5 Klinik fr Viszeral- und Transplantationschirurgie, Hannover Medical School, Hannover, Germany Key words: expression pattern – hCD59-transgenic pigs – health status – phenotype – xenotransplantation Abbreviations: CMV: cytomegalovirus; DAF: decay accelerating factor; DEPC: diethylpyrocarbonate; EF1a: elongation factor 1 alpha; F1, F2: generation number of transgenic offspring; FFPE: formalinefixed paraffine-embedded; hCD59: human complement regulator CD59; Ln./Lnn.: lymph node (lymphonodus)/lymph nodes (lymphonodi); MCP: membrane cofactor protein; PCR: polymerase chain reaction; PCV: porcine circovirus; PRRSV: porcine respiratory and reproduction syndrome virus; RT-PCR: reverse transcriptase-polymerase chain reaction; SPF: specific pathogen free; TNF: tumor necrosis factor. Address reprint requests to Professor Oliver Bock MD, Institute of Pathology, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany (E-mail: [email protected]) or Professor Heiner Niemann, Dept. Biotechnology, Institute of Animal Breeding, (FAL) Mariensee, 31535 Neustadt, Germany (E-mail: [email protected]) Received 25 September 2005; Accepted 9 April 2006 Introduction The critical shortage of human donor organs has prompted considerable efforts in establishing porcine to human xenotransplantation. The first immunological barrier in xenotransplantation is the hyperacute rejection response (HAR), which is mediated by preformed xenoreactive antibodies that are directed against the a-galactosyl-epitope [1,2]. These antibodies activate the complement 345 Deppenmeier et al. cascade of the organ recipient which results in a rapid and irreversible destruction of the xenogenic donor organ [3,4]. Transgenic overexpression of human complement regulators such as CD59, CD55 [decay accelerating factor (DAF)] or CD46 [membrane cofactor protein (MCP)] has been shown to inhibit the complement cascade and reliably prevents HAR [see 5–8]. The complement regulatory protein human complement regulator CD59 (hCD59) inhibits formation of the membrane attack complex at the end of the complement cascade [6–8]. Microinjection of foreign DNA into pronuclei of zygotes has been the method of choice for the production of transgenic domestic animals. However, the technology is associated with a random integration of the transgene and frequently an unpredictable expression pattern in the transgenic animals [9]. Due to the random integration and variable expression patterns, insertional mutagenesis and unwanted pathological side effects cannot be ruled out and concerns have been raised about animal welfare of animals resulting from this approach [10,11]. Ectopic transgene expression has been detected in some cases, but without any obvious pathology [12,13]. Pathomorphological phenotypes have been shown in transgenic and non-transgenic offspring derived from microinjection, in vitro fertilization and/or somatic cloning. Overexpression of various growth hormone (GH) constructs in transgenic pigs or sheep was associated with a specific pathological phenotype, which was avoided by improved GH constructs [14,15]. The large offspring syndrome [16], and specific cardiopulmonary and placental lesions [17–19] and lesions in the skeletal and muscle system [20,21] have been observed as long-term consequences of in vitro handling of bovine and ovine embryos. However, a systematic analysis of potential pathological side effects putatively associated with the random integration and expression of a specific transgene in transgenic domestic animals has not yet been reported. Here, we evaluated the health status of pigs transgenic for the human complement regulator protein CD59 and conducted a complete pathomorphological examination on transgenic and wildtype control animals. The transgenic pigs used in the present study were hemizygous F1–F2 offspring from nine transgenic lines. The founder animals had been produced by pronuclear microinjection of human regulator of complement activation (RCA) constructs into porcine zygotes [22]. The main goals of the present study were to determine (i) the health status of hCD59-transgenic pigs by a detailed pathomorphological analysis, and (ii) if transgenesis is correlated with specific 346 pathological changes in F1–F2 transgenic offspring. In addition, the expression pattern of both hCD59 mRNA and hCD59 protein in a large panel of organs and tissues was determined. Materials and methods Animals Nineteen German Landrace F1 and F2 hCD59transgenic pigs (TG) from nine transgenic lines and nine non-transgenic control pigs (WT), between 1 and 32 months of age were slaughtered at the Institute for Animal Breeding, Mariensee. Both groups were kept under identical specific pathogen free (SPF) conditions. All animals were treated according to institutional guidelines. The sex, age and breeding line of the animals examined are shown in Table 1. We selected a representative number of animals from a total of nine different transgenic lines with a rather broad range in age for this study. This should render the results representative for other pig populations derived from this transgenic approach. The vast majority of the animals analyzed in this study were from cytomegalovirus promoter (CMV)hCD59 transgenic lines; in addition one animal with a CMV-DAF transgene (from line 13) was analyzed. The transgene expression patterns for line 5, carrying a construct in which the CMV promoter (PCMV) drives expression of human CD59, have been characterized previously [22]. Recently, transgenic expression patterns of five lines (13, 17, 19, 20 and 21), carrying a tetracycline responsive promoter (PtTA) have been described [23]. The expression pattern from two lines (8 and 10) with a CMV driven construct had not been published earlier. All animals did not show any obvious health problems. One animal (TG26) was from a double transgenic background (hCD59 · hCD55). Pathomorphological analysis All pigs were killed and organs and tissues were evaluated macroscopically for pathomorphological changes. Lung and Lymphonodus pulmonalis were tested by PCR for porcine circovirus type 2 (PCV 2) and for porcine respiratory and reproductive syndrome virus (PRRSV) by a certified laboratory (Laboklin, Bad Kissingen, Germany). These viruses were included as they are emerging pathogens that are now frequently found in domestic pig populations. Histopathological examination showed evidence (intracytoplasmic, basophilic inclusion bodies in lymph nodes) for a PCV 2 infection in the animals. The human complement regulatory protein hCD59 Table 1. Age, sex and transgenic construct in pigs analyzed for pathologies Animal no. WT1 WT2 WT3 WT4 WT5 WT6 WT7 WT8 WT9 TG6 TG7 TG8 TG1 TG4 TG15 TG20 TG2 TG5 TG23 TG13 TG14 TG19 TG18 TG21 TG25 TG27 TG12 TG26 Age (months) Sex Construct Breeding line 8 12 15 4 18 17 21 28 1 18 4 4 21 7 32 25 17 8 24 7 7 15 11 16 12 1 7 1 Male Male Male Male Female Female Male Male Male Male Female Female Female Male Female Male Female Female Male Male Male Male Male Female Female Male Male Female – – – – – – – – – PCMV CD59 PCMV CD59 PCMV CD59 PCMV CD59-GFP PCMV CD59-GFP PCMV CD59-GFP PCMV CD59-GFP PCMV CD59-GFP PCMV CD59-GFP PtTA DAF-TA PtTA CD59-TA PtTA CD59-TA PtTA CD59-TA PtTA CD59-TA PtTA CD59-TA PtTA CD59-TA PtTA CD59-TA PtTA CD59-TA PtTA CD59-TA PtTA DAF-TA – – – – – – – – – 5 5 5 8 8 8 8 10 10 13 17 19 19 20 20 20 20 21 15 · 20 Litter size Stillborn G TG m TG f 8 (4m, 4f) 11 (3m, 8f) Sib. of TG7 10 (6m, 4f) 13 (7m, 6f) 10 (4m, 6f) Sib. of TG4 10 (3m, 7f) 11 (5m, 6f) – 1 1 1 2 – 2 – – F1 F2 F2 F1 F1 F1 F1 F1 F1 4 2 2 1 2 3 2 2 1 4 6 6 2 2 2 2 2 4 GFP, enhanced green fluorescent protein; Sib, Sibling; G, generation; TG m, male transgenic piglets; TG f, female transgenic piglets; m, male; f, female. The following organs and tissues were fixed in 10% buffered formalin: skin (inner tight), skeletal muscle (M. gracilis), heart (left ventricle, right ventricle, septum), lung (Lobus cranialis, Lobus caudalis), spleen, liver, kidney, urinary bladder, pancreas, brain (cerebellum, cerebrum), thyroid gland, lymphatic nodes (Ln. pulmonalis, Ln. cervicalis superficialis, Lnn. ileofemorales, Lnn. mesenteriales), esophagus, stomach, duodenum, jejeunum, ileum, colon, and aorta. If the organs and tissues did not show macroscopic lesions, samples were taken from representative sites for histopathological examination. If macroscopic lesions were visible, the samples were taken from the site of the lesion. After 24 h of fixation, samples were paraffin-embedded, stained with hematoxylin–eosin and histopathologically evaluated. Determination of integration of the hCD59 construct DNA extraction was performed as described previously [24]. In brief, eight to 10 formalin-fixed, paraffin-embedded (FFPE) samples derived from heart, lung, spleen, liver, kidney, pancreas, brain, skeletal muscle, and esophagus were employed for genomic DNA isolation. The isolated DNA was then adjusted to a final concentration of 50 ng/ll. Primers for amplification of hCD59 DNA were based on the sequence of the hCD59 construct as follows: hCD59 forward: 5¢-GGTCATAGCCTGCAGTGCTACA-3¢, hCD59 reverse: 5¢-AAATCAGATGAACAATTGACGGC-3¢. Primers for hCD59 generated a 77 bp product. The sequence of tumor necrosis factor (TNF) was chosen as control gene to prove successful extraction of genomic DNA. The following TNFspecific primers were employed (GenBank X54001): TNF forward: 5¢-ATAGTATGAGGAGGACTCGCTGAGC-3¢ TNF reverse: 5¢-ACATCCCATCTGTCCATGAGGTT-3¢. The TNF primers generated a 94 bp product. PCR amplification was performed in a GeneAmp PCR System 2700 (Applied Biosystems, Darmstadt, Germany) with a final reaction mixture of 25 ll containing 250 nm of each primer, 0.5 units of Platinum Taq Polymerase (Invitrogen, Karlsruhe, Germany), 200 lm each of dATP, dCTP, dTTP, and dGTP in 1x Platinum Taq reaction buffer with 1.5 mm magnesium chloride and 5.0 ll 347 Deppenmeier et al. DNA. The reaction mixture was preheated at 95C for 5 min, followed by 40 cycles at 95, 60, and 72C, 30 s each. Final extension was performed for 5 min at 72C. DNA derived from fibroblasts transfected with the hCD59 construct served as positive controls. DNA derived from organs and tissues of nontransgenic control pigs and mock PCR (without template) served as negative controls. Analysis of hCD59 mRNA expression Formalin-fixed, paraffin-embedded samples derived from heart, lung, spleen, liver, kidney, pancreas, brain, skeletal muscle, and esophagus of the 19 RCA-transgenic pigs were analyzed. For RNA extraction from FFPE samples, a one-step protocol was used as described previously [25]. Briefly, eight to 10 sections (15 lm each) were cut from the paraffin block with a microtome and placed directly into a 1.5 ml screw cap-tube; 750 ll digestion solution (4 m guanidinium isothiocyanate/0.75 m sodium citrate/10% sarcosyl), 5.25 ll 0.1 b-mercaptoethanol (Merck, Darmstadt, Germany) and 300 ll proteinase K solution (20 mg/ml in H2O; Merck) were added, and the tubes were placed in a thermoshaker for overnight incubation (55C, 1400 g). The following day, the samples were centrifuged at 4C for 5 min at 14 000 g, and 1 ml of each digested sample was transferred into a 2.0 ml tube. After addition of 100 ll 3 m sodium acetate (pH 5.2), 630 ll water-saturated phenol (pH 4.5 to 5.5), and 270 ll chloroform, the sample was mixed thoroughly and stored on ice for 15 min. After centrifugation at 4C for 20 to 25 min at 14 000 g, the upper aqueous phase (approximately 1 ml) was pipetted into a fresh 2.0 ml tube; 1 ll glycogen (20 mg/ml; Roche Molecular Diagnostics, Mannheim, Germany) as an RNA carrier and 1 ml 2-propanol were added to the ribonucleic acidcontaining aqueous phase. After mixing, the samples were stored for at least 24 h at )20C. After precipitation, the samples were centrifuged at 4C for 20 min at 14 000 g. The RNA pellet was washed with 70% ice-cold ethanol, air-dried, and dissolved in 35 ll precooled DEPC-treated H2O. The RNA concentration was then measured spectrophotometrically. Total RNA (1 lg), pretreated with RNase) DNase (1 U/lg RNA; RQ1, Promega, Madison, WI, USA), was then transcribed into complementary DNA using 250 ng random hexamers (Amersham Pharmacia, Freiburg, Germany) and 200 U of SuperScript II Rnase) Reverse Transcriptase (Invitrogen) in a volume of 20 ll following the manufacturer’s protocol. Negative controls were 348 performed by adding water instead of Reverse Transcriptase. Primers used for the hCD59 RT-PCR were identical to the hCD59 DNA PCR (see above). The housekeeping gene elongation factor 1 (EF1) a was chosen as a positive control [26]. The following sequences were used (GenBank XM203909): EF1 a forward: 5¢-CAAAAAY(C/T)GACCCACCAATGG-3¢ EF1 a reverse: 5¢-GGCCTGGATGGTTCAGGATA-3¢. The EF1 a RT-PCR generated a 68 bp product. RT-PCR conditions were similar to the hCD59 PCR as described above. RNA extracted from human kidney samples served as positive controls. RNA derived from various organs and tissues of non-transgenic control pigs, of a hCD55-transgenic pig, and samples without reverse transcriptase ()RT) served as negative controls. Immunohistochemistry for determination of hCD59 protein expression Immunohistochemistry was performed using a tissue microarray technology as previously described [27] employing the same FFPE samples as for molecular analysis. For immunohistochemical staining the tyramine amplification technique was used as described [28]. Slides were deparaffinized, dehydrated and rinsed in deionized water. Antigen retrieval was carried out by incubation with protease XIV (21.4 mg/50 ml aqua dest; SigmaAldrich, München, Germany) for 10 min. Endogenous peroxidase blocking was performed with 3% H2O2 (pH 7.5). Blocking of unspecific binding was achieved by incubating with 0.5% casein for 10 min. To avoid unspecific binding to endogenous biotin an incubation with avidin (Avidin/Biotin blocking kit; Vector, Burlingame, CA, USA) for 10 min each was performed. To detect expression of hCD59, a monoclonal mouse-anti-human CD59-antibody [clone p282 (H19); BD PharMingen, Heidelberg, Germany], diluted 1 : 200 was applied and incubated overnight at 4C. As secondary antibody, biotinylated goat-anti-mouse-antibody (Zytomed, Berlin, Germany) was applied in dilution 1 : 900 for 30 min, followed by horseradish peroxidase complex (Zytomed) diluted 1 : 500 was applied for 30 min. To amplify the reaction, biotinylated tyramine with 0.003% H2O2 was applied for 10 min. Biotinylation of tyramine was performed as reported previously [27]. Incubation with avidin-biotinylated alkaline phosphatase (Zytomed) diluted 1 : 300 was performed for 30 min. The human complement regulatory protein hCD59 For color development, slides were incubated with the chromogen fast red (1 mg fast red salt/1 ml fast red buffer; Sigma-Aldrich) for 8 min, counterstained with hemalum (dilution 1 : 3; Merck) and thoroughly washed with buffer (0.1 m Tris–HCl, pH 7.6/0.15 m NaCl/0.05% Tween 20) after each incubation step. The staining pattern was evaluated and the percentage of positively labeled cells and their distribution pattern was assessed () ¼ negative, + ¼ weak staining in up to 30% of the cells, ++ ¼ moderate staining of 30% to 60% of the cells, +++ ¼ strong staining in >60% of the cells). Human tonsillar tissue served as positive control, whereas tissue derived from non-transgenic pigs served as negative controls. To ensure that the hCD59 staining pattern as determined by screening of tissue arrays represented the true overall staining pattern in transgenic animals, whole tissue sections were stained and evaluated accordingly in positive cases. Results Pathomorphological evaluation of transgenic pigs lines expressing hCD59 Transgenic animals as well as non-transgenic control pigs only rarely showed any pathological lesions. Only in individual animals non-specific pathologies were detected (Fig. 1). One non-transgenic animal (WT3) had a bilateral, abdominal cryptorchism. Another non-transgenic animal (WT7) showed a moderate, focal, chronic, purulent bronchopneumonia with multifocal minerali- zation in the right frontal lobe of the lung. One transgenic animal (TG4) had a severe, focal, chronic, ulcerative gastritis in the pars proventricularis and a severe, multifocal, follicular hyperplasia of peribronchiolar follicles. One transgenic animal (TG7) showed a severe, diffuse, chronic, fibrous epi- and pericarditis and a mild, multifocal, chronic, fibrous pleuritis in the lung. One transgenic animal (TG15) had a moderate, focal, chronic fibrosis of the liver capsule, and transgenic animal TG20 showed histologically multifocal, acute, single cell necroses of hepatocytes. One transgenic animal (TG23) had multifocal, chronic ossification in the mesenterium. In addition to these specific lesions in individual animals, several non-transgenic and transgenic animals showed mild lesions in a variety of organs (Table 2, Fig. 1) including mild, perivascular infiltration in the skin, mild infiltration in the liver, mild, chronic interstitial nephritis, mild infection with Balantidium coli and deposition of siderin containing macrophages in the Lnn. ileofemorales. Nonspecific pathological signs were found in nontransgenic control animals and transgenic animals at the same ratio and to the same extent, and no specific transgene related pathomorphology was apparent. PCV 2 could be detected in 65% of the transgenic animals and 75% of the non-transgenic animals. However, despite the existence of intracytoplasmic, basophilic inclusion bodies in some lymph nodes, no pathomorphological phenotype typical for PCV 2-associated diseases, such as postweaning multisystemic wastening syndrome Fig. 1. Examples of histopathological lesions in transgenic and non-transgenic animals. (A) Lnn. ileofemorales (WT6): Deposition of siderin-containing macrophages (arrow) in the lymph node; (B) Skin (WT7): mild, multifocal, perivascular infiltration with lymphocytes, plasma cells and single neutrophils (arrow); (C) Lung (WT7): moderate, focal, chronic, purulent bronchopneumonia with multifocal mineralization (arrow); (D) Colon (TG7): Balantidium coli (arrow) in the lumen (bar 100 lm). 349 Deppenmeier et al. Table 2. Lesions found in several animals Organ Skin Histopathological lesion Affected animals Affected animals (%) Spleen Mild, multifocal, perivascular infiltration with lymphocytes, plasma cells and single neutrophils Mild, multifocal, infiltration with lymphocytes, macrophages and single neutrophils Mild, multifocal, chronic, non-suppurative, interstitial nephritis Balantidium coli in the lumen without inflammatory reaction Mild to moderate, multifocal, follicular hyperplasia WT1, WT2, WT6, WT7 TG1, TG2, TG5, TG15, TG18, TG19, TG20, TG21 WT3, WT4, WT6, WT7, WT8 TG7, TG8, TG12, TG13, TG14, TG15, TG18, TG19, TG20, TG21, TG25 WT7 TG13, TG20, TG21 WT2 TG5, TG6, TG7, TG18 WT3 44% 47% 56% 58% 11% 16% 11% 21% 11% WT TG WT TG WT TG WT TG WT Ln. pulmonalis Multifocal, acute hemorrhages WT6, WT7 TG18, TG19, TG22, TG23, TG26 WT1, WT2, WT8 TG1, TG2, TG4, TG5, TG13, TG14, TG23, TG26 WT4, WT6, WT7 TG5, TG7, TG8, TG13, TG15, TG18, TG19, TG21 WT2, WT3, WT4, WT5, WT6, WT8 TG4, TG5, TG7, TG12, TG13, TG15, TG18, TG19, TG20, TG21, TG23, TG25 WT4, WT7, WT8, WT9 TG4, TG8, TG13, TG14, TG18, TG19, TG21, TG25, TG26 WT2, WT4 TG4, TG12, TG13, TG14, TG20 WT2, WT3, WT5, WT6, WT7, WT9 TG4, TG5, TG6, TG7, TG8, TG13, TG14, TG26, TG27 WT3, WT5, WT6, WT7, WT9 TG7, TG8, TG15, TG19, TG20, TG23, TG26, TG27 WT2 TG4 WT2, WT4 TG5, TG7, TG8 22% 26% 33% 42% 33% 42% 67% 63% 44% 47% 22% 26% 66% 47% 56% 42% 11% 11% 22% 16% WT TG WT TG WT TG WT TG WT TG WT TG WT TG WT TG WT TG WT TG Liver Kidney Colon Follicular hyperplasia Edema and sinusoidal histiocytosis Lnn. mesenteriales Follicular hyperplasia Edema and sinusoidal histiocytosis Lnn. ileofemorales Follicular hyperplasia Edema and sinusoidal histiocytosis Siderin containing macrophages in the sinus Ln. cervicalis cran. superf. Follicular hyperplasia Edema and sinusoidal histiocytosis (PMWS) or porcine dermatitis and nephropathy syndrome (PDNS) were detected. PRRSV could not be detected in any of the transgenic or control animals. Expression patterns of hCD59 mRNA in organs and tissues of transgenic pigs In general, the previously reported mosaic expression patterns were confirmed [22,23]. Three animals (TG18, TG19, TG25) expressed hCD59 mRNA in all organs examined. Twelve animals (TG1, TG2, TG7, TG8, TG12, TG13, TG14, TG15, TG20, TG21, TG26, T27) expressed hCD59 mRNA only in a subset of organs and tissues. The level of hCD59 mRNA expression varied in organs and tissues within a single transgenic animal as well as between different animals (Table 4, Fig. 2). In four animals (TG4, TG5, TG6, TG23) no hCD59 mRNA expression could be detected. The hCD59 mRNA was consistently demonstrated in positive controls whereas in negative controls and samples without reverse transcriptase ()RT) hCD59 mRNA expression was never determined. EF1 a transcripts could be detected in all samples indicating 350 successful RNA extraction, cDNA synthesis, and RT-PCR. Samples without )RT were predominantly negative, but some cases showed weak false positive amplification products, probably due to an incomplete DNase digestion. As expected, hCD59 DNA could be detected in all animals derived from the group of hCD59transgenic animals (data not shown), confirming transmission of the transgene over several generations. Non-transgenic animals were negative for hCD59-DNA. Organ- and tissue-specific expression patterns of hCD59 protein in transgenic animals Thirteen of the 19 transgenic pigs showed cellular expression of the hCD59 protein in various organs and tissues (Table 3, Fig. 3). From these 13 animals, none showed a consistent expression pattern of the transgene in all organs. Cellular hCD59 protein expression could be demonstrated only in a subset of organs and tissues, and with remarkable variations between different animals. In some animals a ‘‘patchy’’ expression pattern was detected which was characterized by distinct positive labeling of single cells in heart and kidney, 21 15 · 20 20 13 17 19 10 8 5 ++ End. + End. ) + End. ) ) +++ End. ) ) ) ) ) ++ End. +++ End. ) ) ) ) ) Skin ++ End. + Card. ++ ) + End. ++ End. + End. ) ) ) ) ) ++ End. + End. ) ) ) ) ) Heart Spleen + End. ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) Lung ) ++ Alv., End. ++ Alv., End. ++ Alv., End. ++ Alv., End ++ Alv., End. ++ Alv., End. ) ) ) ) ) +++ Alv., End. +++ Alv., End. ) ) ) ) ) + End. ) + End. ) ) ) ) ) ) ) ) ) ++ End. ) ) ) ) ) ) Liver ) ) ) ++ End. ) ++ End. + End. ) ) ) ) ) + End. + End. ) ) ) ) ) Kidney + End. ) ) ) ++ End. ) ) ) ) ) ) ) ) ) ) ) ) ) ) Urinary bladder +++ Acin. En. ) + Acin. ) ) + Acin. ) ) ) ) ) ) ) ) ) ) ) ) ) Pancreas Number and intensity of positively staining cells (referring to the tissue in general): +++ strong, ++ moderate, + weak, ) negative. End., endothelial cells; Card., cardiomyocytes; Alv., alveolar epithelial cells; Ac., acinic cells; En., endocrine pancreas (islet cells); Myoc., myocytes. TG 6 (hCD59) TG 7 (hCD59) TG 8 (hCD59) TG1 (hCD59) TG 4 (hCD59) TG 15 (hCD59) TG 20 (hCD59) TG2 (hCD59) TG5 (hCD59) TG23 (hCD55) TG13 (hCD59) TG14 (hCD59) TG19 (hCD59) TG18 (hCD59) TG21 (hCD59) TG25 (hCD59) TG27 (hCD59) TG12 (hCD59) TG26 (hCD59+hCD55) L. Table 3. Pattern of hCD59 protein expression in transgenic pigs ++ End. ) ++ End. ) ) +++ End. ) ) ) ) ) ) ) ) ) ) ) ) ) Brain ) ) ) ) ) ++ End. ) ) ) ) ) ) + Myoc. ++ Myoc. ++ Myoc. + Myoc. + Myoc. ) + Myoc. Skeletal muscle ++ End. ) ++ End. ) + End. ) ) ) ) ) ) ) ) ++ Myoc. ++ Myoc. + Myoc. + Myoc. ) + Myoc. Esophagus ) ) + End. ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) Duodenum + End. ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) Jejunum ) ) ++ End. ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) Ileum ++ End. ) ++ End. ) ++ End. ) ) ) ) ) ) ) ) ++ End. ) ) ) ) ) Colon The human complement regulatory protein hCD59 351 Deppenmeier et al. Table 4. Comparative analysis of hCD59 protein and mRNA expression in transgenic pigs Heart TG6 TG7 TG8 TG1 TG4 TG15 TG20 TG2 TG5 TG23 TG13 TG14 TG19 TG18 TG21 TG25 TG27 TG12 TG26 5 5 5 8 8 8 8 10 10 13 17 19 19 20 20 20 20 21 15 20 Lung Spleen Liver Kidney Pancreas Skeletal muscle Brain Esophagus Prot. RNA Prot. RNA Prot. RNA Prot. RNA Prot. RNA Prot. RNA Prot. RNA Prot. RNA Prot. RNA ++ + ++ ) + ++ + ) ) ) ) ) ++ + ) ) ) ) ) ) ++(+) ) ) ) ++ ++ ) ) ) ) ) ++(+) +++ + ++ ) ) ) ) ++ ++ ++ ++ ++ ++ ) ) ) ) ) +++ +++ ) ) ) ) ) ) ++ (+) + ) ) ++ + ) ) ) ) ) +++ ++ ++ ++ +(+) + ) + ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) + ) ++ +++ ++ ) ) ) ++ +++ ++ ++(+) ++ ) +(+) ) + ) + ) ) ) ) ) ) ) ) ) ++ ) ) ) ) ) ) ) ++ ++ ) ) ) ) ) ) ) ) ++ +++ ++ (+) +++ ) ) ) ) ) ) ++ ) ++ + ) ) ) ) ) + + ) ) ) ) ) ) ) ) +++ ) ) +++ ) ) ) + ++ +++ ++ ++(+) +++ ) + ) ) + ) ) ) ) ) ) ) ) ) ) ) ) ) ) n.d. ) ) ) ) ) ) ) ) + + +(+) ++ ) ++ ) ) ) ++ ) ++ ) ) +++ ) ) ) ) ) ) ) ) ) ) ) ) ) ) n.d. ++ ) ) + + ) ) ) ) + +++ +(+) ) n.d. ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) + ++ ++ + + ) + ) ) ) ) ) ) + ) ) ) + ) +++ +++ +++ +++ ++ ) +(+) ++ ) ++ ) + ) ) ) ) ) ) ) ) ++ ++ + + ) + ) ) ) ) ) ++ ++ ) ) ) + ) +++ +++ ++(+) +++ + ) + ++ + ) n.d., not done. +++ strong, ++(+) strong to moderate, ++ moderate, +(+) weak to moderate, + weak, ) negative. Fig. 2. Variable hCD59 mRNA expression as demonstrated for a hCD59 transgenic animal. (A) hCD59 mRNA could be detected by RT-PCR in heart, lung, spleen, kidney, brain, muscle and esophagus whereas liver and pancreas virtually showed no expression of the transgene. hCD59-transfected cells served as the positive control. Non-transgenic pig without expression. (B) RT-PCR detection of EF1 a-transcripts. with neighboring cells remaining negative for the hCD59 protein (Fig. 3). The following organs showed a positive, intracytoplasmic staining for 352 hCD59 protein: skin, heart, lung, spleen, liver, kidney, urinary bladder, skeletal muscle, esophagus, duodenum, jejunum, ileum and colon. Endo- The human complement regulatory protein hCD59 Fig. 3. Representative staining pattern of hCD59 protein in a variety of organs derived from different transgenic animals. (A) Kidney (TG1): expression in glomerular endothelial cells (arrow) and in the endothelial cells of an afferent arteriole (arrowhead); (B) Lung (TG8): expression in alveolar epithelial cells type I (arrowhead) and type II (arrow); (C) Heart (TG7): expression in the cytoplasm of a cardiomyocyte (arrow) without staining of the neighboring cells, ‘‘patchy pattern’’; (D) Pancreas (TG6): expression in acinary cells of the exocrine pancreas and in single islet cells (arrows); (E) Pancreas (TG15): expression in acinary cells of the exocrine pancreas (arrow), ‘‘patchy pattern’’, no staining of endothelial cells; (F) Heart (TG6): expression in endothelial cells (arrow); (G) Brain (TG15): expression in endothelial cells (arrow); (H) Skin (TG6): expression in endothelial cells (arrow); (I) human tonsillar tissue used as positive control: expression in endothelial cells; (J–O) negative controls of a non-transgenic pig (WT4): (J) kidney; (K) lung; (L) heart; (M) pancreas; (N) brain; (O) skin. A, D, I, J, M, O: ·250; B, C, E, F, G, H, K, L, N: ·400 (bar 100 lm). thelial cells of small and medium blood vessels expressed hCD59 protein. In the lung, not only endothelial cells, but also alveolarepithelial cells type I and II showed hCD59 labeling. In the pancreas, acinic cells of the exocrine pancreas, and islet cells of the endocrine pancreas showed hCD59 353 Deppenmeier et al. protein expression. The acinic cells of the exocrine pancreas of one animal (TG15) showed a patchy expression pattern. The cytoplasm, especially the outer membrane, of some cardiomyocytes in animal TG7 stained patchy for hCD59. The myocytes in the skeletal muscle and the muscular layer of the esophagus in six animals (TG18, TG19, TG21, TG25, TG26, TG27) showed a patchy expression pattern of hCD59, with only a part of the skeletal muscle cells showing a positive reaction. In the kidney of one animal (TG1), endothelial cells of the capillaries in the glomerula and afferent arteries showed a positive staining, with only single glomerula showing the positive reaction. Non-transgenic animals never showed expression of hCD59protein. Discussion The study reported here is the first systemic analysis to detect pathological side effects potentially associated with expression of a specific transgene, using hCD59 as a model. Results show that in transgenic pigs produced by microinjection of foreign DNA into pronuclei the health status is not different from that of the wild-type control animals. A total of 19 transgenic pigs from nine lines were analyzed. As the production and breeding of transgenic pigs is a time consuming, complex and costly process, we restricted the number of transgenic animals to be analyzed to one to four animals per transgenic line and covered a rather broad range of different ages. This approach ensures representative results also for other transgenic programs. Wild-type animals from the same facility served as controls. It is well known that random integration of a foreign DNA could be associated with insertional mutagenesis [9]. A major prerequisite for a potential usage of transgenic porcine organs in xenotransplantation is that animal health and welfare are not compromised. Here, we show that microinjection of foreign DNA is not associated with a specific pathomorphological phenotype in pigs. Some of the animals had chronic interstitial nephritis, pneumonia or epi- and pericarditis. These lesions are common pathological features especially in older animals in modern pig populations and were probably caused by previous viral or bacterial infections [29]. Because of the chronic nature of these lesions, the causative agent could not be detected any more. Lesions in organs like kidney or lung show that it is of crucial importance to check the organs for transferable diseases before xenotransplantation. The activation of spleen and lymph nodes as shown by follicular hyperplasia, 354 edema and sinusoidal histiocytosis, can be attributed to a previous challenge with unspecific pathogens. As the animals were not maintained under gnotobiotic conditions, but kept under SPF conditions, a challenge with ubiquitous pathogens is likely to occur. The deposition of siderin-containing macrophages in the sinus of the ileofemoral lymph nodes are likely remnants from ferric injections which are commonly performed in pig populations. Non-transgenic control animals and transgenic animals were affected in approximately the same ratio and to the same extent by the lesions (Table 2). Prior to the use of porcine organs in xenotransplantation, the donor animals must be screened thoroughly for possible infections with pathogens, because of the risk of transmitting these pathogens to the host [5]. In the majority of the transgenic and nontransgenic animals PCV 2 was detected by PCR. PCV 2 is found widely within modern pig populations and in most cases a PCV 2 infection is not associated with clinical or pathomorphological changes. In some cases the infection can be associated with certain diseases, including PMWS or PDNS [30]. In this study, none of the PCV IIpositive transgenic and non-transgenic pigs showed any signs of such PCV II-associated diseases. In accordance with the original report a varying expression pattern for hCD59 was detected in lines 5, 17, 19, 20 and 21. Lines 17, 19, 20 and 21 showed a muscle fiber-confined expression of hCD59. In some cases the mRNA, but not the protein could be detected. This might be due to a higher sensitivity of the PCR assay compared with the immunohistology. CD59 mRNA was variably expressed in the tested animals. In few animals mRNA expression of the transgene was not detected (Table 4). As hCD59 protein was detected in heart, lung, and skin of these animals (Table 3), this most likely reflects RNA degradation in these samples. Similar to the hCD59 mRNA, the protein expression pattern was variable as well. Despite the use of an ubiquitously active promoter (CMV) no animal expressed the protein in all organs, instead most animals expressed the transgene in a subset of organs. In addition, a patchy expression pattern could be detected in heart and kidney. A likely explanation for the variable expression pattern could be the promoter and/or the impact of chromosomal flanking regions. The CMV promoter had been selected because it is known to induce a strong and ubiquitous transgenic expression in cell lines and transgenic mice as well [31,32]. However, reports from different transgenic animal models confirm our finding that CMV and The human complement regulatory protein hCD59 CMV-derived promoter expression can vary both within different lines and even between tissues of a single line, and can thus result in mosaic patterns even within one tissue [33–36]. Recent studies and own results point to de novo DNA methylation in transgenic pigs as a mechanism to silence expression [23,37]. Crossbreeding experiments of lines 13 and 20 resulted in a broad tissue-unspecific expression of hCD59 [23] highlighting that breeding of the existing lines might yield pigs with an ubiquitous expression of human complement regulators. In summary, no evidence for pathomorphological changes was found in a representative number of transgenic pigs from nine transgenic pig lines of the F1 and F2 generation, including animals with high levels of the transgenic expression in several tissues. 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Epigenetic regulation of lentiviral transgene vectors in a large animal model. Mol Ther 2006; 13: 59. Arch Virol (2006) DOI 10.1007/s00705-006-0868-y Printed in The Netherlands Brief Report Inhibition of porcine endogenous retroviruses (PERVs) in primary porcine cells by RNA interference using lentiviral vectors B. Dieckhoff1; , A. Karlas1; , A. Hofmann2 , W. A. Kues3 , B. Petersen3 , A. Pfeifer2, H. Niemann3 , R. Kurth1 , and J. Denner1 1 Robert Koch-Institute, Berlin, Germany Department of Pharmacy, Ludwig-Maximilians-University, M€ unchen, Germany 3 Department of Biotechnology, Institute for Animal Science, Neustadt, Germany 2 Received July 14, 2006; accepted September 13, 2006; published online November 16, 2006 # Springer-Verlag 2006 Summary A potential risk in pig-to-human xenotransplantation is the transmission of PERVs to human recipients. Here we show for the first time the inhibition of PERV expression in primary porcine cells by RNA interference using lentiviral vectors. Cells were transduced with lentiviral vectors coding for short hairpin (sh) RNAs directed against PERV. In all primary porcine cells studied and in the porcine kidney cell line PK-15, expression of PERV-mRNA was significantly reduced as measured by real-time PCR. Most importantly, expression of PERV proteins was almost completely suppressed, as shown by Western blot analysis. Thus, lentiviral shRNA vectors could be used to knockdown PERV expression and create transgenic pigs with a reduced risk of PERV transmission during xenotransplantation. The transplantation of porcine xenografts may offer a potential way to overcome the shortage of Authors contributed equally. Author’s address: Dr. Joachim Denner, Robert Koch-Institute, Nordufer 20, 13353 Berlin, Germany. e-mail: [email protected] allogeneic organs. However, xenotransplantation may be associated with the risk of transmission of PERVs, which are integrated in the porcine genome [3, 23] and have been found to be released as infectious particles from normal pig cells [15, 17, 26]. Three subtypes of PERVs (A, B and C) are known which mainly differ in the env region encoding for the receptor-binding site [12, 23, 29]. PERV-A and PERV-B are polytropic viruses and able to infect human cells in vitro [16, 22, 25, 29, 30], whereas PERV-C is an ecotropic virus. Even though PERVs are able to infect human cells in vitro, no virus transmission has been observed in previous experimental and clinical xenotransplantations [6, 8, 21, 27]. However, in most of these approaches, the time of contact was short and the immunosuppression was weak. In contrast, the intended conditions for future xenotransplantations include long-term transplantation of porcine cells or organs, direct contact of human and porcine cells, and a strong immunosuppression to avoid rejection. PERV transmission has also not been observed in pig-to-nonhuman primate transplantations and infection experiments [2, 14, 31]. However, the risk of PERV transmission associated with tumours or immunodeficiencies is still existent. Therefore, different strategies to B. Dieckhoff et al. prevent PERV transmission have been developed, such as the selection of low-producer animals [20, 28], the generation of an antiviral vaccine [4], and the inhibition of PERV expression by RNA interference [9, 18]. In these first experiments, inhibition of PERV expression by small interfering RNAs (siRNAs) was studied in PERV-infected human cells. In this study, we describe for the first time inhibition of PERV expression in primary porcine cells transduced by lentiviral vectors expressing antiPERV shRNA. We studied the genetic distribution, the expression pattern of different PERV subtypes, and the inhibitory effect of two different lentiviral vectors on PERV expression. In order to analyze the presence of PERV-A, PERV-B and PERV-C proviruses, DNA was isolated from three foetal fibroblast cultures (SE101, SE105 and P1F10) obtained from individual D25 (p.c.) German landrace foetuses [11] as well as from PK-15 cells, using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany), and a PCR was Fig. 1. Analysis of PERV provirus integration and expression in porcine primary fibroblasts and PK-15 cells. A Detection of PERV proviruses in the genome of two independent primary fibroblast cultures (SE101, P1 F10) and PK-15 cells using PCR. B PERV expression in foetal fibroblasts (SE101 and P1 F10) and in PK-15 cells using onestep RT-PCR. In addition to specific primers for env-A, env-B, env-C, primers specifically detecting full-length and spliced env mRNA were used. Integrity of RNA was determined by amplifying GAPDH performed using primers specific for env of subtypes A, B [12] and C [29]. All proviruses were found integrated in the genome of the primary fibroblasts P1 F10 and SE101, whereas PK-15 cells did not contain PERV C (Fig. 1A). In order to study PERV expression, total RNA was isolated (RNeasy Kit, Qiagen), and a onestep reverse transcription (RT) PCR (Invitrogen, Karlsruhe, Germany) was performed using the same primers as described above as well as primers allowing the discrimination between viral fulllength RNA (forward: 50 TGCTGTTTGCATCAA GACCGC, reverse: 50 ACAGACACTCAGAA CAGAGAC) and spliced env-mRNA (forward: 50 TGCTGTTTGCATCAAGACCGC, reverse: 50 ATGGAGGCGAAGCTTAAGGGGA). The integrity of the RNA was determined by amplifying the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH, for: 50 CTGCCCCTT CTG CTGATGC; rev: 50 TCC ACGATGCCGAA GTTGTC). In all primary cells tested, full-length mRNAs corresponding to the PERV subtypes A and B and C as well as spliced env mRNA were detected (Fig. 1B). Since no PERV-C proviruses were integrated in PK-15 cells, no PERV-C-specific mRNA was found (Fig. 1B). The presence of spliced env mRNA indicates that Env protein may be generated and consequently virus particles may be produced. In our previous publications, different synthetic siRNAs corresponding to several parts of the sequence of PERV were designed and screened for their ability to inhibit expression of PERV in infected human 293 cells [9, 10]. The most effective siRNA corresponded to a highly conserved sequence in the pol gene (pol2); the target sequence is identical in all functional PERV strains. Like other siRNA used, the pol2 siRNA was able to inhibit PERV-A and PERV-B [10]. Inserted into the vector pSuper, permanent expression of this polspecific shRNA was achieved, and efficient inhibition of PERV expression was observed [9, 10]. This pSuper-pol2 construct was chosen as a basis for establishing a lentiviral expression system. Two different lentiviral vectors, RRL-pGK-GFP [5] (kindly provided by L. Naldini) and pLVTHM [32] (kindly provided by D. Trono), which allow expression of the corresponding short hairpin Inhibition of PERV expression by RNA interference Fig. 2. Schematic representation of the lentiviral vectors RRL-pGK-GFP and pLVTHM. LTR long terminal repeat, H1 polymerase III H1-RNA gene promotor, shRNA short hairpin RNA, SIN self-inactivating element, cPPT central polypurine tract, GFP green fluorescent protein, WPRE post-transcriptional element of woodchuck hepatitis virus, EF-1alfa elongation factor-1a promoter, teto tetracycline operator, hPGK phosphoglycerate kinase promotor, loxP locus of X-over of P1, recombination site of Cre recombinase for bacteriophage P1 RNAs, were used (Fig. 2). They differed in the promoters inducing the expression of the GFP reporter (pLVTHM used an EF1a promoter, whereas RRL-pGK-GFP used a PGK promoter) and in the arrangement of shRNA and GFP expression cassettes. In the case of the pLVTHM, the shRNAexpressing cassette is located within the 30 -LTR (long terminal repeat). The shRNA expression cassette of the vector pSuper-pol2 was inserted into pLVTHM and RRL-pGK-GFP using the restriction enzymes EcoRI and ClaI, and EcoRI and HindIII (New England Biolab, Frankfurt, Germany), respectively, resulting in pLVTHM-pol2 and RRLpGK-GFP-pol2. Lentiviral particles were obtained by co-transfecting the packaging cell line 293T with the envelope plasmid pCL-VSV-G, the packaging plasmid psPAX2, the transfer vector pLVTHM, and the transfer vector pLVTHM-pol2. Titration of concentrated lentiviral particles was performed on Hela cells as described (lentiweb.com). The titres were 1.73108 IU=ml for pLVTHM and 1.78 107 IU=ml for pLVTHM-pol2. RRL-pGK-GFPpol2 and RRL-pGK-GFP were produced as described recently [7, 24]. The titres were 2.25 1010 and 2.01010 IU=ml, respectively. For transduction of foetal fibroblasts and PK-15 cells, 1105 cells were seeded in a 6-well culture plate in the presence of 6 mg=ml polybrene in DMEM and incubated with concentrated lentiviral particles at a MOI of 2.5 for 5 days. By fluorescence microscopy and flow cytometry, transduction efficiencies of 90% were measured. In the case of lower efficiencies, transduced cells were enriched by cell sorting (MoFlo, Dako, Glostrup, Denmark) or repeated addition of lentiviral particles. As a result, transduction efficiencies of around 95% were achieved. Total RNA and protein were isolated from transduced and non-transduced foetal fibroblasts and PK-15 cells using TRI Reagent (Sigma, Steinheim, Germany) and the RNeasy Kit (Qiagen). To measure PERV expression quantitatively, a SYBR Green real-time RT-PCR was performed as described previously [9]. An inhibition of PERV mRNA expression of 82.5 and 78% was observed in foetal fibroblasts SE101 and SE105 using the vector RRLPGK-GFP-pol2 (Fig. 3A). In addition, a one-step RT real-time PCR (Invitrogen) was performed using a FAM-labelled probe (50 FAM-AGAAGG GACCTTGGCAGACTTTCT [3BQ1], Sigma), primers specific for PERV gag (forward: 50 TCCAGGGCTCATAATTTGTC, reverse: 50 TGA TGGCCATCCAACATCGA), and the Mx4000 multiplex quantitative PCR system (Stratagene, La Jolla, USA). The assay revealed a coefficient of correlation of 0.980 with an almost optimal efficiency of amplification. For each reaction, 50 ng total RNA was used. The amount of full-length RNA was calculated relative to the constituted amount of total RNA using a modification of the 2 CT method [13], and the expression of PERV in cells transduced with shRNA lentiviral particles was compared with the expression in cells transduced with the control vector as well as non-transduced cells. An inhibition of PERV full-length mRNA expression of 81.8% was observed in foetal fibroblasts P1 F10 transduced with pLVTHM-pol2 (Fig. 3A). When P1 F10 cells that had been sorted for GFP-positive cells in order to increase the number of transduced cells (nearly 99%) were analysed, the inhibition of PERV expression increased up to 94.8%. Inhibition of PERV expression in trans- B. Dieckhoff et al. duced cells was stable and remained at 95% over months (Fig. 3A). Finally, it is important to note that expression of PERV in primary fibroblasts was very low (0.2%) when compared with the expression in virus-producing PK-15 cells (defined as 100%). After showing that pLVTHM-pol2 and RRLPGK-GFP-pol2 were able to inhibit PERV expression in foetal fibroblasts, their effect in another cell type was studied. Epithelial PK-15 cells were transduced with both lentiviral vectors, RRL-PGK-GFPpol2 and pLVTHM-pol2, at an efficiency of 95% and showed an inhibited PERV expression of 65.8 and 73.5%, respectively (Fig. 3B), indicating that PERV expression can be inhibited in different cell types. PERV expression was also reduced in PHAtreated PBMCs, which were difficult to analyse since the transduction efficiency was relatively low. However, after enrichment of GFP-expressing PBMCs by fluorescence-activated cell sorting, an inhibition of PERV full-length mRNA expression of 75.2% was observed. In order to analyse whether reduced expression of PERV mRNA leads to a reduced expression of viral proteins, protein samples of foetal fibroblasts P1 F10 and PK-15 cells were analysed using a Western blot assay. Analysing protein lysates of transduced and non-transduced PK-15 cells (50 mg) by Western blot with a goat anti-p27Gag-antiserum [8], a reduced PERV expression was detected in cells transduced with both lentiviral pol2 vectors (Fig. 3C). Loading of equal protein amounts was verified using antibodies against b-actin (Sigma). However, even if 80 mg total protein lysate of fetal fibroblasts P1 F10 was used, no Gag protein was detected even in the non-transduced control cells, 1 Fig. 3. Expression of PERV in cells transduced with the lentiviral vectors RRL-PGK-GFP-pol2 and pLVTHM-pol2 and in cells transduced with control vector as well as in non-transduced cells (set 100%). A Three batches of foetal fibroblasts and B PK-15 cells transduced with both vectors were analysed at day 5 or at other time points after transduction using one-step RT real-time PCR. C Western blots showing expression of p27Gag in PK-15 cells transduced with RRL-PGK-GFP-pol2 and pLVTHM-pol2 and in controls. D RT activity in the supernatants of PK-15 cells transduced with RRL-PGK-GFP-pol2 and pLVTHM-pol2, and control cells Inhibition of PERV expression by RNA interference suggesting that the PERV protein expression in these cells is under the detection level. To analyse whether viral reverse transcriptase (RT) activity was released into the supernatants of control and transduced foetal fibroblasts P1 F10 and PK-15 cells, an RT activity assay was performed (Cavidi Bio Tech, Uppsala, Sweden). Transduced and control PK-15 cells were seeded at a density of 5104 cells in 24-well plates and RT activity was measured in the supernatants after three days. This assay demonstrated a reduced RT activity in the supernatants of PK-15 cells transduced with both lentiviral vectors, RRL-PGK-GFP-pol2 (5.78 mU=ml) and pLVTHM-pol2 (4.74 mU=ml), compared with the expression of untreated control cells (21.21 mU=ml) (Fig. 3D). In the supernatants of fetal fibroblasts P1 F10, no RT activity (0 mU=ml) was detected, confirming the low expression of PERV even in untreated cells. In this study, we show for the first time a significant inhibition of PERV expression in primary porcine cells by lentiviral vector-mediated RNA interference. These data correlate with previous experiments investigating PERV-specific RNA interference in human cells infected with PERV-B or with a recombinant PERV-A=C using synthetic siRNAs and the pSuper vector system [9, 10, 18]. Lentiviral vectors were chosen, firstly, to increase the transduction efficiency, and secondly, because these vectors can be used to generate transgenic pigs by injecting them into the perivitelline space of porcine embryos [7], or transfected primary cells can be employed in somatic nuclear transfer [19]. Expression of shRNA depends on the number of integrated vectors per cell and on the promotor activity. Although the expression cassette of pLVTHM-pol2 is doubled after lentiviral integration [32], no significant difference in the knockdown efficiency could be detected compared to RRL-pGK-GFP-pol2 (Fig. 3A, B). Due to the strong polymerase III H1-RNA gene promoter, which allows ubiquitous expression of shRNAs, a single shRNA expression cassette per lentiviral vector might be sufficient to express enough shRNAs to reduce the PERV mRNA to a minimum. Moreover, after integration of the lentiviral vector into the host genome, the knockdown of PERV expres- sion was stable in primary porcine cells for more than 170 days (Fig. 3A). Little is known about the expression of PERV in different tissues and organs of normal and transgenic pigs [1]. The data presented here suggest that the expression of PERV may be inhibited in different cell types including blood cells, which is important for the use of cells and organs for xenotransplantation. 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Kues, Heiner Niemann * Department of Biotechnology, Institute for Animal Breeding, Mariensee, 31535 Neustadt, Germany Received 10 April 2007 Available online 7 May 2007 Abstract In mice hemizygous for the Oct4-GFP transgene, the F1 embryos show parent-of-origin dependent expression of the marker gene. F1 embryos with a maternally derived OG2 allele (OG2mat/) express GFP in the oocyte and during preimplantation development until the blastocyst stage indicating a maternal and embryonic expression pattern. F1-embryos with a paternally inherited OG2 allele (OG2pat/) express GFP from the 4- to 8-cell stage onwards showing only embryonic expression. This allows to study allele specific knock down of GFP expression. RNA interference (RNAi) was highly efficient in embryos with the paternally inherited GFP allele, whereas embryos with the maternally inherited GFP allele showed a delayed and less stringent suppression, indicating that the initial levels of the target transcript and the half life of the protein affect RNAi efficacy. RT-PCR analysis revealed only minimum of GFP mRNA. These results have implications for studies of gene silencing in mammalian embryos. 2007 Elsevier Inc. All rights reserved. Keywords: Oct4-GFP transgene; RNA interference; Maternal inheritance; Paternal inheritance; Short interfering RNA; Preimplantation development; Gene expression Development of early mammalian embryos is characterized by a switch from a maternal to an embryonic expression program [1]. Maternally derived transcripts and proteins are deposited in the oocyte and consumed after fertilisation at least until the embryonic genome is activated. The RNA binding protein Msy2 has been proposed to stabilize maternal mRNAs in the oocyte [2]. The timing of the major onset of embryonic genome transcription is species specific. In mouse embryos it occurs at the 2-cell stage, in human embryos at the 4-cell stage, and in bovine embryos at 8–16-cell stage. A ‘‘minor’’ genome activation has been observed prior to the major genomic activation. Some genes show a biphasic expression pattern, in that maternally derived mRNAs and proteins are present in the oocyte and that the gene are also actively transcribed after embryonic activation. It has been shown that RNA interference (RNAi)-mediated gene knock down works in mammalian embryos for several genes including E-cadherin, Mos, Plat, Oct4, * Corresponding author. Fax: +49 5034 871 101. E-mail address: [email protected] (H. Niemann). 0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.04.155 Cox5a, Cox5b or Cox6b1 [3–9]. However, it is unknown whether RNAi is equally effective for maternal and embryonic transcripts. Maternal transcripts have a long half-life in the range of days, whereas embryonic transcripts last at most for minutes. In the present study, a breeding schedule was applied to produce hemizygous embryos carrying a marker transgene that permits a comparison of gene silencing of the same allele expressed either maternally or after embryonic genome activation. The zygotes analysed in this study were hemizygous for a GFP transgene driven by the promoter of the POU transcription factor Oct4 [10,11]. It has been previously shown that the Oct4 promoter activates transcription in early blastomeres from the 4- to 8-cell stage onward, in the inner cell mass (ICM) of preimplantation embryos, in the epiblast and eventually in the gametes [12]. The Oct4-GFP transgene has been shown to be active in oocytes and both mRNA and GFP protein are maternally stored in MII oocytes and zygotes. Terminally differentiated spermatozoa, however, do not express Oct4. Mating of homozygous OG2 males or females with non-transgenic mice, produced hemizygous OG2 F1 embryos with either embryonic GFP 728 K. Iqbal et al. / Biochemical and Biophysical Research Communications 358 (2007) 727–732 expression after the 4-cell stage (OG2pat/), or zygotes with maternal deposits of GFP mRNA and protein (OG2mat/), i.e. maternal expression. Thus knock down of one of two alleles of the target gene located at the same integration site, sharing identical regulatory and structural elements, and identical secondary structure of the transcripts could be studied in two different modes of expression. Materials and methods Production of zygotes hemizygous for the OG2 transgene. OG2 transgenic mice (tg; Oct4 promoter-GFP) [10,11] were maintained as an inbred homozygous line by sib matings. For the production of hemizygous OG2 zygotes, OG2 animals were mated with non-transgenic NMRI animals. OG2 and NMRI females were superovulated by intraperitoneal injections of 10 IU PMSG (pregnant mare serum gonadotropin, Intervet, Unterschleißheim, Germany), followed by injections of 10 IU hCG (human chorion gonadotropin, Intervet) 48 h later and mating with OG2 or NMRI males, respectively. Zygotes were collected by flushing the oviducts of plugged females 22–24 h after hCG injection with phosphate buffered saline (PBS) containing 0.3% (w/v) bovine serum albumin (BSA). Cumulus cells were completely removed by a short treatment with 0.03% hyaluronidase and zygotes were collected in PBS/0.3% BSA on a warming plate. Animals were maintained and handled according to German guidelines. Injection of siRNA and in vitro culture. The lyophylised siRNAs were dissolved at a concentration of 20 lM in a buffer consisting of 30 mM HEPES/KOH (pH 7.4), 100 mM K-acetate, and 2 mM Mg-acetate and stored frozen at 20 C in 10 ll aliquots. Denuded zygotes were washed in PBS/0.3% BSA and transferred to a micromanipulation unit in a droplet of PBS/0.3% BSA. Individual zygotes were fixed by suction to a holding capillary, approximately 10 pl of siRNA solution was injected into the cytoplasm using an Eppendorf transjector 5246 (Eppendorf, Hamburg, Germany). Rhodamine conjugated siRNAs (Qiagen, Hilden, Germany) with 3 0 -TT overhangs against GFP (upper strand: 5 0 -GCAAGCUGA CCCUGAAGUUCAU) or with a random sequence (upper strand: 5 0 -UU CUCCGAACGUGUCACGU) with no known homology to mammalian genes were used for injection. Successful injections were verified by fluorescence microscopy. Fertilized embryos of both inheritance patterns were microinjected with either the GFP-siRNA or the control random-siRNA, untreated embryos were used as culture controls. Groups of 10 embryos were cultured for 4.5 days in 50 ll microdrops of KSOM medium overlaid with silicone oil at 37 C in an atmosphere of 5% CO2 in air. For fluorescence microscopy, a Zeiss Axiovert 35 M microscope equipped with fluorescence optics for Hoechst 33342, GFP and rhodamine was used [13]. Images were recorded on Ektachrome 320 T film (Kodak) with a Contax167MT camera. The GFP fluorescence signals were normalized by using a fixed exposure time of 15 s. 4-cell stage embryos and blastocysts were collected for RT-PCR and stored frozen at 80 C. At the end of the in vitro culture period developmental stage, nuclei number and GFP mRNA levels were determined. For determination of the number of nuclei per blastocyst, embryos were stained with 1 lg/ml Hoechst 33342 for 5 min. Determination of GFP transcript levels by RT-PCR. Poly(A)+ RNA was isolated from single embryos using a Dynabeads mRNA DIRECT Kit (Dynal, Norway, Product number 610.11) as described [14]. The mRNAs were reverse transcribed in a reaction mixture consisting of 1· RT buffer (50 mM KCl, 10 mM Tris–HCl, pH 8.3, Perkin-Elmer), 50 mM MgCl2, 1 mM dNTPs (Amersham, Brunswick, Germany), 20 IU RNase inhibitor (Perkin-Elmer) and 50 U/ll reverse transcriptase (RT, PerkinElmer) in a total volume of 20 ll using 2.5 ll of 50 lM random hexamer primers (Perkin-Elmer Vaterstetten, Germany). The RT reaction was carried out at 25 C for 10 min and then 42 C for 1 h followed by denaturation at 99 C for 5 min. The polymerase chain reaction (PCR) was performed with the reverse transcribed product using 0.2 embryo equivalents. The final volume of the PCR mixture was 50 ll (1· PCR buffer, 20 mM Tris buffer–HCl (pH 8.4), 1.5 mM MgCl2, 200 lM dNTPs, 0.25 lM of each primer). The GFP primers were 5 0 -AGC ACG ACT TCT TCA AGT CC and 5 0 -TGA AGT TCA CCT TGA TGC CG (annealing temperature: 58 C, 35 cycles). For poly(A) polymerase (also known as Papola; Mouse Nomenclature Page, www.informatics.jax.org/mgihome) amplification, the primers were 5 0 -CCT CAC TGC ACA ATA TGC CGT CTT CAC CTG and 5 0 -AGC CAA TCC ACT GTT CTC CCA CCT TTA CTC (annealing temperature 54 C, 35 cycles). The PCR cycle numbers were optimized to ensure that the reactions are in the linear range of amplification. PCR amplicons were separated by electrophoresis on a 2% agarose gel, and were recorded using a CCD camera (Quantix, Photometrics, Munich Germany). Sequence identities of amplicons were identified by sequencing. Results Hemizygous embryos with a maternally inherited Oct4GFP allele (OG2mat/) expressed GFP in oocytes and zygotes; while embryos with a paternally inherited Oct4GFP allele initiated transcription of GFP after embryonic activation and displayed GFP fluorescence at the 4–8-cell stage (Fig. 1A). Pronuclear stage zygotes of both inheritance patterns were used for siRNA injection, the siRNAs were conjugated with rhodamine, thus successful injection and metabolism of the siRNAs could be followed by fluorescence microscopy (Fig. 1B and C). The majority of embryos had reached the blastocyst stage at the end of the culture period at day 4.5. Apparently the injection procedure did not reduce the blastocyst rate compared to the untreated control group (Table 1). After injection of GFP-siRNA, green GFP fluorescence was significantly suppressed in embryos with paternally inherited Oct4-GFP during the entire observation period (Fig. 2A) and 95% of blastocysts showed down-regulation of GFP fluorescence (Table 1). On average, the GFP mRNA levels were reduced by 97.4% in siRNA treated (OGpat/)-blastocysts as determined by RT-PCR analysis (Fig. 3A and B). In contrast, embryos with a maternally inherited GFP allele (OG2mat/) showed a delayed reduction of GFP fluorescence after siRNA injection. All stages from zygote to morula continued to display GFP fluorescence; a significant knock down of GFP fluorescence became was only apparent at the blastocyst stage (Fig. 2B). The remaining GFP transcript levels in blastocyst stages were low and not different between embryos with a maternally (OGmat/) or a paternal allele (OGpat/) inherited allele (Fig. 3C) indicating that maternal and embryonic transcripts were equally susceptible to RNAi. To determine whether maternally derived GFP transcripts were degraded slower after RNAi injection, OGmat/ zygotes were injected with GFP-siRNA and early 4-cell stages were analysed by RT-PCR. At this time point, embryonic transcription of the Oct4-GFP transgene had yet not started. RT-PCR indicated only minimum levels of GFP transcripts (data not shown), suggesting that predominantly K. Iqbal et al. / Biochemical and Biophysical Research Communications 358 (2007) 727–732 729 Fig. 1. (A) Expression of GFP in F1-embryos with hemizygous OG2 allele from paternal (OG2pat/) or maternal (OG2mat/) inheritance in zygotes, 2cell embryos, morulae and blastocysts. (B) Schematic drawing of rhodamine conjugated siRNA. (C) Injection of siRNA-rhodamine in (OG2mat/) zygotes. Top, brightfield image; middle, GFP fluorescence; bottom, rhodamine fluorescence in injected zygote and injection capillary. Table 1 Knock down of GFP in (OGpat/) and (OGmat/) mouse embryos (OGpat/) GFP-siRNA injected Random-siRNA injected Untreated control (OGmat/) GFP-siRNA injected Untreated control No. of zygotes No. of morulae with knockdown of GFP/total (%) No. of blastocysts with knockdown of GFP/total (%) 210 31 60/172 (93%) 0/25 (0%) 133/140 (95%) 0/19 (0%) 60 0/53 (0%) 0/47 (0%) 94 47 0/79 (0%) 0/39 (0%) 66/66 (100%) 0/35 (0%) the protein turnover time of GFP contributed to the delayed down-regulation. Injection of a random siRNA with no known murine complementary sequence did not affect GFP fluorescence. Rhodamine fluorescence was detected in the cytoplasm of all embryonic stages up to the blastocyst, indicating slow degradation of the synthetic siRNAs (data not shown). Discussion Here, we show that a single injection of a synthetic siRNA into the cytoplasm of mouse zygotes is sufficient to induce allele specific silencing during embryonic development. Employing hemizygous F1 embryos with parent-oforigin dependent expression pattern of the OG2 transgene, allowed discriminating between embryonic and maternal origin of gene expression. Silencing was highly efficient in embryos carrying a paternally inherited GFP allele (OG2pat/). In this setting the siRNA was injected 2–3 cell cycles prior to activation of the GFP allele. Nascent GFP transcripts apparently were targeted and subsequently degraded. The long-term knock down of GFP in OG2pat/ blastocysts up to day 4.5 led us to speculate that the duration of silencing might last to implantation. This suggests that it would be possible to silence genes during the peri- and postimplantation period and to dissect critical pathways in this important period of gestation. Current methodology to target these stages [15–17] is complicated and time consuming. It is not yet known at which developmental stage the unspecific interferon mediated response becomes prevalent, thus the application of siRNAs might be superior to the commonly used long double stranded (ds)RNA. 730 K. Iqbal et al. / Biochemical and Biophysical Research Communications 358 (2007) 727–732 Fig. 2. Knock down of GFP expression in hemizygous embryos with either a paternally or a maternally inherited GFP allele. 2-cell stage, morula and blastocyst stages are shown under brightfield and GFP fluorescence conditions. Fig. 3. RT-PCR detection of GFP mRNA in siRNA injected embryos. (A) OG pat/ embryos: lane 1, untreated embryo; lane 2, untreated, no reverse transcriptase (RT); lane 3, GFP-siRNA injected; lane 4, no RT; M, 100 bp DNA ladder; lane 5, no template. Poly(A) polymerase was amplified as control transcript. (B) OGmat/ embryos: lane 1, GFP-siRNA injected; lane 2, GFP-siRNA injected, no RT; lane 3, untreated embryo; lane 4, untreated embryo, no RT; M, 100 bp ladder; lane 5, no template. (C) Knock down efficiency of GFP transcripts in hemizygous OGpat/ and OGmat/ blastocysts (3 replicates; SD). RNAi was discovered in nematodes and plants [18,19], where long dsRNAs of 500–1000 bp specifically suppress expression of complementary transcripts. In most mammalian cell types, dsRNA causes a non-specific effect mediated by an interferon mediated response [20,21]. This non-specific interferon response is not observed in early embryonic stages of mammals. It has been shown that specific RNA interference can be achieved in mammalian embryos by the injection of long dsRNA [3–6,8]. Processing of the injected dsRNAs by cellular RNaseIII-like endoribonuclease (Dicer) leads to the release of several different short interfering (si)RNAs [22] in the embryo, of which some might be functional as specific RNAi. Commonly, only 1 out of 4 tested siRNAs gives specific gene knock down [23,24]. However, off target effects and siRNA overload [25] cannot be excluded if dsRNA is used for injection into mammalian embryos. K. Iqbal et al. / Biochemical and Biophysical Research Communications 358 (2007) 727–732 An important finding of this study is that the onset and efficiency of gene knock down in embryos critically depend on whether the target gene is expressed from the maternal or embryonic genome. In OG2mat/ embryos, which already carry maternal GFP mRNAs and protein at the time of injection, knock down was delayed and significant GFP fluorescence remained up to the 16-cell stage. Efficient reduction of GFP expression was observed in blastocysts, albeit with higher residual GFP fluorescence than in siRNA treated OG2pat/ embryos. In mouse LA-9 cells the half-life of GFP was determined to be 26 h [26], thus confirming that genes with maternal expression can be silenced, but turnover rates of maternal RNA and protein may delay the inhibitory effects of RNAi. Previously, it has been shown that both maternal and embryonic target genes could be silenced, e.g E-cadherin, Mos, Plat, Oct4, Cox5a, Cox5b or Cox6b1 [3–7]. However, due to the unique orchestration of gene expression in mammalian embryos with a switch from maternal to embryonic expression, RNAi experiments for transcripts with a biphasic expression pattern might be obscured. Phenotypic changes of RNAi in embryos could be masked in cases when proteins with long half lives or slow turnover rates are still active, although transcripts have already been knocked down. The data presented here provide important clues for gene silencing in mammalian preimplantation embryos. The siRNA approach could also be suitable for species other than mouse in which genomic sequencing is not yet complete [27]. Synthesis of siRNAs requires knowledge of short sequence regions and the effects of synthetic siRNA are transient, attributed to degradation of the original molecules, unless transgenic delivery of siRNA is employed [23]. However, it seems to be worthwhile to further investigate the stability of synthetic siRNAs in embryos and to determine their efficacy in pre- and peri-implantation stages. A potential application of synthetic siRNA injection in embryos is the knockdown of gene functions with long term consequences for the developing organism. For example, it has been shown that a specific telomere elongation program acts at morula–blastocyst transition [28], a transient knock down of telomerase in this critical time window should help to elucidate the biological relevance of this mechanism. Acknowledgments The expert technical assistance of E. Lemme is gratefully acknowledged. Khursheed Iqbal is in the Ph.D. program of Hannover Biomedical Research School (HBRS). Khursheed Iqbal was supported by a grant from Goyaike S.A.A.C.I. Y F., Buenos Aires, Argentina. References [1] R.M. Schultz, The molecular foundations of the maternal to zygotic transition in the preimplantation embryo, Hum. Reprod. Update 8 (2002) 323–331. 731 [2] J. Yu, N.B. Hecht, R.M. Schutz, Expression of MSY2 in mouse oocytes and preimplantation embryos, Biol. Reprod. 65 (2001) 1260– 1270. [3] P. Svoboda, P. Stein, H. Hayashi, R.M. Schultz, Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference, Development 127 (2000) 4147–4156. 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USA 101 (2004) 8034–8038. CLONING AND STEM CELLS Volume 9, Number 3, 2007 © Mary Ann Liebert, Inc. DOI: 10.1089/clo.2006.0009 Production of Viable Pigs from Fetal Somatic Stem Cells NADINE HORNEN, WILFRIED A. KUES, JOSEPH W. CARNWATH, ANDREA LUCAS-HAHN, BJÖRN PETERSEN, PETRA HASSEL, and HEINER NIEMANN ABSTRACT Fetal somatic stem cells (FSSCs) are a novel type of somatic stem cells that have recently been discovered in primary fibroblast cultures from pigs and other species. The goal of the present study was to produce viable piglets from FSSCs. NT complexes were prepared from both FSSCs and porcine fetal fibroblasts (pFF) to permit comparison of these two donor cell types. FSSCs from isolated attached colonies were compared with pFF in their ability to form blastocysts upon use in NT. Fusion and cleavage rates were similar between the two groups, while blastocyst rates were significantly higher when using pFF as donor cells. FSSCs of three different size categories derived from dissociation of spheroids yielded similar results. The use of FSSCs of 15–20 m in size yielded similar cleavage and blastocyst rates as fetal fibroblasts. In the final experiment NT complexes produced from FSSCs were transferred to foster mothers. After transfer to prepubertal gilts, three of seven recipients established pregnancies and delivered seven piglets, of which three piglets were viable and showed normal development. Results for the first time demonstrate that FSSCs are able to produce cloned embryos, and that pregnancies can be established and viable piglets can be produced. INTRODUCTION S (NT) is a useful technology for basic research and the production of transgenic animals. NT permits targeted modification of the genome of the donor cells by homologous recombination and production of genetically identical groups of transgenic animals (Denning et al., 2001). Since the first somatic clone “Dolly” (Wilmut et al., 1997), 11 other mammalian species have been cloned successfully by the same technique (Baguisi et al., 1999; Betthauser et al., 2000; Campbell et al., 1996; Cibelli et al., 1998; Chesne et al., 2002; Galli et al., 2003; Lee et al., 2005a; Li et al., 2006; Onishi et al., 2000; Polejaeva et al., 2000; Shin et al., 2002; Wakayama et al., 1998; Woods et al., 2003; Zhou et al., 2003). However, despite intensive efforts OMATIC CELL NUCLEAR TRANSFER cloning, efficiency in most species is still low. In pigs, on average 1%–3% of reconstructed embryos survive to birth (Harrison et al., 2004; Kues and Niemann, 2004). The differentiation status of the donor cells has been assumed to contribute to the success of cloning since correct epigenetic reprogramming and the resulting changes in transcriptional control are the main processes involved in creating an embryo from a somatic nucleus (Jaenisch et al., 2002). Nuclear reprogramming is a physiological process which takes place after fertilization and in early cleavage stages to regulate gene expression during embryogenesis. The reprogramming includes remodeling of the chromatin structure, changes in DNA-methylation, regulation of the expression of imprinted genes, regeneration of telomere length, and inactivation of the X-chromosome (Han et al., Department of Biotechnology, Institut für Tierzucht, Mariensee Neustadt, Germany. 364 365 FSSCS IN SOMATIC CELL NUCLEAR TRANSFER 2003; Westphal, 2005). In somatic cell nuclear transfer the chromatin structure of the differentiated nucleus must be removed and remodeled to initiate the embryogenic process after fusion of the donor cell nucleus with the enucleated oocyte (Gurdon et al., 2003; Westphal, 2005). Presumably, reprogramming to the embryonic state should be completed before the onset of embryonic gene expression (EGA) (Jaenisch et al., 2002). In bovine development, EGA occurs between the 8- and 16-cell stage; in the pig, it occurs at the four-cell stage (Lequarre et al., 2003; MaddoxHyttel et al., 2001). This additional time for remodeling could explain the higher cloning efficiency in bovine nuclear transfer because the transferred nucleus would have more time to be reprogrammed. Stem cells, as well as differentiated cells, have been successfully used as donor cells, at least to the extent that they gave development to the blastocyst stage. Many of these embryos, however, failed to develop further after transfer to foster mothers (Kato et al., 2000; Wakayama and Yanagimachi, 2001). The terminally differentiated cells used include lymphocytes and cardiomyocytes, but all result in rather low efficiency (Hochedlinger and Jaenisch, 2002; Inoue et al., 2005; Schwarzer et al., 2006). In mice the use of embryonic stem cells (ES cells) increased the efficiency from 1%–3% to 15% (offspring/transferred embryos) (Wakayama et al., 1999). True ES cells have not yet been isolated from livestock (Gjorret and Maddox-Hyttel, 2005). Adult stem cells are undifferentiated cells in a differentiated tissue. They are able to proliferate, self-renew, and give rise to differentiated daughter cells. In adult tissue, they are responsible for regeneration and maintainance of homeostasis. Many different types of adult tissue harbor stem cells including bone marrow, brain, epidermis, blood, liver, skin, eye, gut, pancreas, and skeletal muscle (Schoeler, 2004). We have recently described the derivation of fetal somatic stem cells (FSSCs) from explant cultures of various species (Kues et al., 2005). These cells do not show signs of senescence in high density culture, lose fibroblast markers, and express markers of pluri- or totipotency-like OCT4, AP, and STAT3. FSSCs could be more amenable to reprogramming, and would thus be a new source of donor cells in somatic cell nuclear transfer or cell therapy approaches (Kues et al., 2005). However, the isolation and integration into an existing nuclear transfer protocol had not yet been investigated, and was the subject of this research. MATERIALS AND METHODS Cell culture A porcine fetus was obtained by hysterectomy of a pregnant gilt at day 25. It was washed twice in Dulbecco phosphate-buffered saline solution (D-6650, Sigma, St. Louis, MO). Head, legs, and viscera were removed and remaining tissue was sliced into 1–2-mm sections. Tissue slices were placed in drops of recalcified bovine plasma (e.g., 9 parts bovine plasma, P-4639, Sigma (St. Louis, MO) 1 part 0.5 M CaCl2) in a tissue culture flask (#9025, TPP, Switzerland). Supplementation of plasma with calcium is required for fixation of tissue. After agglutination of the plasma drops, DMEM (Dulbecco Modified Eagle Medium) medium was added (supplemented with 2 mM glutamine, 1% nonessential amino acids, 0.1 mM mercaptoethanol, 100 U/mL penicillin, 100 g/mL streptomycin, all from Sigma) 10% fetal calf serum (FCS, Gibco, Karlsruhe, Germany)], and somatic explant cultures were incubated in a humidified 95% air, 5% CO2 atmosphere at 37°C. Outgrowing cells were trypsinized and subpassaged twice prior to cryopreservation. Five to 6 days prior to nuclear transfer, cells FIG. 1. 3D colonies with surrounding monolayer of fibroblasts (original magnification 100). 366 HORNEN ET AL. moved by repeated pipetting. Denuded oocytes were transferred to TL-HEPES covered with mineral oil (Silicone fluid DC200, #35135, Fa. Serva Biochemica, Heidelberg). Nuclear transfer FIG. 2. Floating spheroids in suspension culture (original magnification 100). were thawed and reseeded on tissue culture test plates (92406, Fa. TPP, Switzerland) with DMEM 10% FCS. The cell cycle of the fetal fibroblasts was synchronized in G0/G1 by serum deprivation in DMEM with reduced FCS content (0.5%) 48 h before nuclear transfer. Growth of FSSCs was induced by DMEM culture medium with increased serum content (30%). FSSCs were maintained either as attached cells, in which case 3D colonies formed on the confluent culture (Fig. 1), or in a suspension culture which permits the growth of free floating spheroids (Fig. 2). For nuclear transfer, 3D colonies were isolated from surrounding fibroblasts by aspirating with a 100 L pipette (Fig. 3), floating spheroids were pelleted and washed with PBS. Cells were dissociated by incubation for 10–15 min in 0.025% EDTA/ trypsin, pelleted, washed once in TL-HEPES containing 10% FCS, once in serum-free TL-HEPES, and resuspended in TL-HEPES. Denuded oocytes were enucleated by removing the first polar body and the metaphase plate under UV-light control after Hoechst 33342 staining of the oocytes. A single donor cell was placed into the perivitelline space (Hoelker et al., 2005). Oocyte donor cell complexes were placed between two platinum electrodes and fused with one DC pulse of 1 kV/cm for 99 sec (Multiporator, Eppendorf, Hamburg). Oocyte-donor cell complexes and 50–60 nonmanipulated oocytes (parthenogenetic controls) were activated in an activation chamber (Bügel-Fusionskammer, Fa. Krüss, Hamburg) with one DC pulse of 1 kV/cm for 45 sec (Hoelker et al., 2005). Then the complexes were activated chemically by exposure to NCSU23 with 2 mM 6-dimethylaminopurin for 3 h. Culture of embryos to blastocysts After activation up to 60 reconstructed embryos and parthenogenetic controls were each placed in 500 L NCSU23 in one well of a fourwell dish. Embryos were cultured for 7 days in an incubation chamber (Nuclear Incubation Chamber; Billups-Rothenberg Inc., Del Mar, CA) at 38°C with 5% CO2, 5% O2, and 90% N2. After 7 days of in vitro culture, embryos were stained with 1 g/mL Hoechst 33342 to determine the number of nuclei using a fluorescence microscope. To determine the number of inner cell mass cells and trophectoderm cells, embryos were dif- Collection and culture of cumulus–oocyte complexes Ovaries from prepubertal gilts were collected from local abattoirs. Oocytes with at least three complete layers of cumulus cells were collected and matured in vitro for 40 h (Hoelker et al., 2005). Then, matured oocytes were transferred to TLHEPES supplemented with 0.1% hyaluronidase and incubated for 5 min. Cumulus cells were re- FIG. 3. Isolation of 3D colonies by aspiration. 367 FSSCS IN SOMATIC CELL NUCLEAR TRANSFER ferentially stained. Blastocysts were incubated in a permeabilizing solution containing the ionic detergent Triton X-100 and the fluorochrome propidium iodide to stain trophectoderm cells for 50 sec. Thereafter blastocysts were incubated in a second solution containing 100% ethanol for fixation and the fluorochrome Hoechst 33342 for staining the inner cell mass cells for 4 min. Fixed and stained blastocysts were analyzed with a fluorescence microscope at 100 magnification (Thouas et al., 2001). Embryo transfers Embryos cloned from FSSCs were surgically transferred into the oviducts of gilts that were synchronized to the reconstructed embryos (Walker et al., 2002), immediately after activation. Maintenance of pregnancies was supported by induction of accessory corpora lutea injecting 1000 IU PMSG injected i.m. at day 12 after embryo transfer (Pregnant Mare’s Serum Gonadotropin, Intergonan® i.m., Intervet, Tönisvorst) and 500 IU hCG (human Chorionic Gonadotropin, Ovogest®, Intervet, Tönisvorst) 72 h later. Recipients were examined ultrasonographically at days 22 and 35 following embryo transfer. Piglets were delivered on days 116–119 after embryo transfer. Animal handling was in accordance with institutional guidelines. were compared with fetal fibroblasts as donor cells. In the second experiment, FSSCs of different size categories, that is, 14 m, 15–20 m, 23–28 m, isolated from spheroids were compared. In the third experiment, blastocysts were cloned from medium size FSSCs (15–20 m) derived from spheroids and compared with that of fetal fibroblasts. In the final experiment, cloned zygotes produced from FSSC nuclear donor cells were transferred to foster mothers. Spheroids were not serum deprived prior to use in nuclear transfer. The different cell size of spheroids does not correspond with different cell cycle stages, but is thought to be related to the differentiation status of stem cells. Statistical analysis Fusion rate, the cleavage rate, the blastocyst rate, the number of nuclei in the blastocysts, and the percentage of ICM cells compared to the total cell number of the blastocysts were analyzed by “Sigma Stat” (Jandel Scientific Coorporation, Germany). Results are based on 4–10 replicates per experiment, and are expressed as means (mean SD). A probability value of p 0.05 was considered to be statistically significant. RESULTS Microsatellite analysis Experiment 1 Parentage was analyzed in piglets derived from somatic cell nuclear transfer to confirm genetic identity of the piglets with the donor cells used for somatic cell nuclear transfer. The microsatellite analysis was performed as descibed before (Hoelker et al., 2005). Eleven porcine DNA microsatellite markers were used (S0002, S0068, S0090, S0101, S0178, SW69, SW 632, SW 857, SW 911, SW 951, and SW1122). In the experiment to determine whether FSSCs removed from selected three-dimensional colonies by trypsinization were better nuclear donors than primary fibroblasts, the blastocyst rate of FSSC reconstituted embryos was significantly lower than the blastocyst rate of the fibroblast group (6.4% 3.5% vs. 24.9% 8.6%). Parthenogenetic embryos used as control for development without nuclear transfer gave a blastocyst rate of 44.3% 9.1%. Cleaveage rates and the mean cell number of the blastocysts in all three groups were not different (Table 1). In this experiment, a wide distribution of sizes was observed within the trypsinized population of FSSCs. Experimental design The first three experiments were designed to determine which FSSC preparation was most suitable to nuclear transfer based on in vitro culture to the blastocyst stage. The assay was to compare the development of these FSSC derived embryos to that of embryos reconstructed from porcine fetal fibroblasts which our laboratory uses routinely for nuclear transfer. In the first experiment, FSSCs from isolated attached colonies Experiment 2 In this experiment to determine whether the size of FSSC donor cells was an important factor, FSSCs of different sizes were isolated from spher- 368 HORNEN ET AL. TABLE 1. BLASTOCYST RATES, CLEAVAGE RATES, AND MEAN CELL NUMBER OF BLASTOCYSTS OF EMBRYOS DERIVED FROM 3D COLONIES AND FETAL FIBROBLASTS Experimental groups FSSCs Fetal fibroblasts Parthenogenetic ap n Cleavage rate (%) Blastocyst rate (%) Cell number of blastocysts (mean SD) 138 166 356 64.8 17.3 82.5 5.60 81.6 11.7 6.4 3.5a 24.9 8.6b 44.3 9.1b 32 80 31 13 32 11 0.05, six replications. oids and used as donor cells. A comparison of the blastocyst rates, cleavage rates, and mean cell number from the three different cell sizes and for the parthenogenetic controls is shown in Table 2. There was no significant difference which could be attributed to the size of the donor cells and the blastocyst rate for the parthenogenetic controls was normal (33.2% 8.8%). Experiment 3 In the experiment to determine whether FSSC donor cells derived from spheroids were better or worse than fibroblasts, there was no significant difference between the two cell types with respect to blastocyst rate, cleavage rate, or mean number of cells in the blastocysts (Table 3). Differential staining, revealed no significant differences between the two cell types with respect to the number of ICM cells, the number of TE cells, total number of cells, or the number of cells in the ICM as a proportion of the total cell number (Table 4). Experiment 4 In this experiment to evaluate the quality of reconstituted embryos by their capacity for in vivo development, embryos cloned from FSSCs were transferred to synchronized foster mothers, and at 25 days three of the seven recipients were pregnant. A second ultrasound examination at 32 days showed that these three animals were still pregTABLE 2. nant. The eighth recipient died shortly after surgery for reasons unrelated to the experiment and was not included in the analysis. The three sows which remained pregnant delivered a total of seven female piglets. Three of the piglets survived and showed normal development (Figs. 4 and 5). Microsatellite analysis confirmed that the piglets had been derived from the FSSCs. The other four piglets had normal birthweights and no obvious deformaties. A detailed pathological examination suggested that they had developed normally. DISCUSSION Successful nuclear transfer depends on correct and complete epigenetic reprogramming of the donor cell nucleus to convert the genome of a differentiated somatic cell into the totipotent state (Gurdon and Colman, 1999). It has been suggested that a less differentiated nuclear donor cell could be more efficiently reprogrammed (Jaenisch et al., 2002) and, indeed, in the mouse, cloning from pluripotent embryonic stem cells resulted in higher rates of development than cloning with differentiated donor cells (Wakayama et al., 1999). Embryonic stem cells have not yet been isolated from livestock species; however, we recently reported a cell culture system that enriches a subpopulation of FSSCs in the pig, the BLASTOCYST RATES, CLEAVAGE RATES, AND MEAN CELL NUMBER OF EMBRYOS RECONSTRUCTED WITH SPHEROID FSSCS OF DIFFERENT SIZE CLASSES Experimental groups Small’ FSSCs Medium’ FSSCs Large’ FSSCs Parthenogenetic controls Five replications. n 145 135 142 259 Cleavage rate (%) 83.5 83.1 83.2 88.8 7.8 1.6 5.8 8.9 Blastocyst rate (%) 15.3 17.6 10.4 33.2 7.9 6.8 2.7 8.8 Cell number of blastocysts (mean SD) 32 34 31 34 13 13 16 11 369 FSSCS IN SOMATIC CELL NUCLEAR TRANSFER TABLE 3. BLASTOCYST RATES, CLEAVAGE RATES, AND MEAN CELL NUMBER OF BLASTOCYSTS OF EMBRYOS DERIVED FROM SPHEROID FSSCS AND FETAL FIBROBLASTS n Cleavage rate (%) Blastocyst rate (%) Cell number of blastocysts (mean SD) 348 322 586 80.7 5.9 86.1 6.7 84.2 7.3 18.4 5.6 21.4 5.6 36.1 5.8 35 11 37 13 34 11 Experimental groups FSSCs Fetal fibroblasts Parthenogenetic Ten replications. cow, and the mouse. These FSSCs exhibit many of the characteristics of pluripotent ES cells (Kues et al., 2005), including absence of senescence in a high-density culture, loss of typical fibroblast markers, and expression of typical stem cell markers such as OCT4, AP, and STAT3. Injection of mouse FSSCs into the blastocoel resulted in chimerism in fetal mesenchymal organs, and occasionally labeled cells were identified in the genital ridge (Kues et al., 2005). We hypothesized that FSSCs would require less reprogramming than more differentiated cell types, and could evolve as a novel source of donor cells in somatic nuclear transfer. In the present study, we report the birth of the first cloned piglets from somatic stem cells and demonstrate the capacity of FSSCs for in vivo development. After transferring 70–100 reconstituted embryos to each recipient, three of seven gilts (42.9%) became pregnant. Harrison et al. (2004) reported a higher efficiency with fetal fibroblasts and recent experience in our laboratory is similar. An early observation of the FSSC population in culture revealed different cellular morphologies depending on culture conditions. Before going directly to the in vivo experiments, it was necessary to evaluate the developmental competence of these different classes of FSSC. The FSSCs could be grown as three dimensional colonies by allowing them to grow first as a high density culture attached to a dish. The FSSCs could also be grown as spheroid cultures by suspension culTABLE 4. ture. In both cases it was necessary to trypsinize the cell aggregates to obtain single cells for nuclear transfer, and in both cases, the trypsinization resulted in a range of cell sizes. In removing three-dimensional colonies from the culture dish prior to EDTA/trypsin treatment, it was impossible to avoid some degree of contamination by the fibroblasts which form a monolayer on the surface of the dish and provide a substrate for colony growth. Trypsinization of these colonies released some fibroblasts into the single cell suspension which must be avoided when the cells were to be used as nuclear donor cells. Spheroid cultures did not have this disadvantage, and thus were expected to provide pure FSSC samples. Another problem with using the three-dimensional cultures as sources of donor cells was the extended period of exposure to the EDTA/trypsin treatment which was necessary to give single-cell suspensions. Indeed, one explanation for the low blastocyst rate in the first experiment could be attributed to the stress of trypsin isolation on these donor cells. This idea is supported by the fact that many damaged cells could be observed in these preparations. In subsequent experiments, in addition to changing to suspension cultured FSSC cells, we also were able to shorten the EDTA/trypsin treatment. When using fetal fibroblasts as NT donor cells, it is desirable to select small round cells which have been demonstrated to be typical for the G0/G1 phase of the cell cycle (Boquest et al., 1999). Fetal fibroblast cultures have a greater RESULTS OF DIFFERENTIAL STAINING OF THE BLASTOCYSTS FSSCS OR FETAL FIBROBLASTS AS DONOR CELLS WITH Experimental groups FSSCs Fetal fibroblasts Six replications. Blastocysts n Number of ICM cells (mean SD) Number of TE cells (mean SD) Total cell number (m) ICM cells/total cell number (%) 24 25 16 5 17 8 22 12 24 10 39 14 39 11 41.7 14.3 42.3 17.3 370 HORNEN ET AL. FIG. 4. The three vital piglets 1 day after birth. number of cells with this favoured morphology after 48 h of serum deprivation. Unfortunately, cultures of FSSCs, both 3D colonies and spheroids, are heterogeneous with regard to cell size, and it is necessary to make a choice when selecting cells for nuclear donation. The FSSCs of “medium” size corresponded to the size of the fetal fibroblasts. Interestingly, the results of the second experiment showed no significant difference in development which could be related to donor cell size. This indicates that cell size is not an appropriate selection criterion for NT with FSSCs. Having determined that a more homogeneous and more viable population of FSSCs could be isolated from suspension produced spheroids and having learned that cell size was not an important trait for selection, the next step was to directly compare fibroblasts and FSSCs with respect to the developmental potential of embryos produced from these two types of nuclear donor cells. It is obvious that medium-size FSSCs isolated from spheroids have the same developmental potential after nuclear transfer as fetal fibroblasts (20%). For comparison, the blastocyst rates reported with various types of differentiated porcine nuclear donor cells range from 10.3% to 37% (Boquest et al., 2002; Hyun et al., 2003; Kim et al., 2006; Lee et al., 2003a, 2003b, 2005b, 2005c). It is difficult to compare the results from different laboratories due to the many methodological differences with regard oocyte source (in vivo vs. in vitro maturation, young or adult sows, slaughterhouse derived or fresh ovaries, etc.). In some instances, there is no visual control of enucleation success, which means that enucleation may not have been achieved in all instances (Boquest et al., 2002; Hyun et al., 2003; Lee et al., 2003b). Lee et al. (2003b) injected donor cells into the cytoplasm of oocytes and obtained a blastocyst rate of 37%. Other laboratories have looked at various types of less differentiated donor cells. For example, dedifferentiated preadipocytes gave a blastocyst rate of 20.8% (Tomii et al., 2005). Zhu et al. (2004) obtained blastocyst rates of 25% with porcine skin-derived stem cells (PSOS), Bosch et al. (2006) used porcine mesenchymal stem cells (MSC) as donors and reported a blastocyst rate of 4.1% and Sung et al. (2006) used long-term and short-term repopulating hematopoietic stem cells and hematopoietic progenitor cells as donors and reported morula/blastocyst rates of 4.1%–10.6%. The present experiments used a nuclear transfer protocol which had been optimized for fetal fibroblasts. FSSCs are different from fibroblasts in that they exhibit several different morphologies depending on culture conditions. This presumably reflects physiological differences as well, so it is likely that a nuclear transfer protocol specifically optimized for FSSC would give increased the cloning efficiency. Comparison of the quality of fetal fibroblast and FSSC derived cloned embryos (i.e., total cell number and the ratio of ICM cells to the total cell number) showed that there was no significant difference between the two types of donor cell. The total cell numbers for both groups were similar to cell counts previously reported (Park et al., 2001a, 2001b, 2001c, 2002). In general, total cell numbers of in vivo produced blastocysts or IVP FIG. 5. Piglets at age of 2.5 months. 371 FSSCS IN SOMATIC CELL NUCLEAR TRANSFER embryos are higher than those reported for cloned embryos. This may be due to the fact that optimal culture conditions have not yet been developed for cloned porcine embryos (Niemann and Rath, 2001). Indeed, the current status of porcine preimplantation embryo culture is far behind that of bovine or mouse embryo culture where significantly higher blastocyst rates are routine. We have achieved a degree of improvement in that the proportion of ICM cells to the total number of cells in the blastocysts was higher than previously reported for cloned embryos (Koo et al., 2004). Normal fetal development requires an adequate number of both ICM and TE cells for implantation (Tam, 1988). Bovine cloned blastocysts often show a higher total cell number and a higher proportion of ICM cells than IVF or in vivo developed embryos (Koo et al., 2002). A distortion in this ratio can be associated with malformations of the placenta and early fetal loss (Koo et al., 2002). In our in vivo experiment, we did not observe malformations of the placentas or abortions. In contrast to bovine cloning experiments, the number of TE cells was normal and the number of ICM cells was increased (Koo et al., 2004). The increase of ICM cells was observed for both FSSCs and fetal fibroblasts. These embryos still do not match the total cell number or the ICM/TE ratios of normal embryos developing in vivo, but the increase in ICM cells indicates that progress is being made toward developing an optimized somatic nuclear transfer protocol. Stem cell markers including Oct4 and alkaline phosphatase (AP) should facilitate separation of FSSC from contaminating fibroblasts (Yeom et al., 1996; Kues et al., 2005). One possibility for improving the selection of FSSC donor cells could be to transfect the population with a gene construct consisting of Oct4 as a promotor and an appropriate flurorescent reporter gene or even an antibiotic resistance gene. This would enable one to isolate pure FSSCs with high efficiency. Another possibility would be the use of antibodies against stem cell specific surface markers either to visualize FSSCs or to purify them by FACS. Human CD133 (formerly AC133) is one such pluripotent stem cells marker (Miraglia et al., 1997; Yin et al., 1997); however, its presence on FSSCs has not yet been shown. In conclusion, results of this study show that FSSCs have at least the same in vitro developmental potential as fetal fibroblasts using an established nuclear transfer protocol and are able to generate vital piglets following embryo transfer. Improved selection and handling of the FSSCs could increase the efficiency. ACKNOWLEDGMENTS This work was supported by the H. 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Zhu, H., Craig, J.A., Dyce, P.W., et al. (2004). Embryos derived from porcine skin-derived stem cells exhibit enhanced preimplantation development. Biol. Reprod. 71, 1890–1897. Address reprint requests to: Heiner Niemann Department of Biotechnology Institut für Tierzucht D-31535 Neustadt, Germany E-mail: [email protected] CSIRO PUBLISHING Reproduction, Fertility and Development, 2007, 19, 762–770 www.publish.csiro.au/journals/rfd Transgenic farm animals: an update Heiner NiemannA,B and Wilfried A. KuesA A Department of Biotechnology, Institute for Animal Breeding, Mariensee, 31535 Neustadt, Germany. author. Email: [email protected] B Corresponding Abstract. The first transgenic livestock species were reported in 1985. Since then microinjection of foreign DNA into pronuclei of zygotes has been the method of choice. It is now being replaced by more efficient protocols based on somatic nuclear transfer that also permit targeted genetic modifications. Lentiviral vectors and small interfering ribonucleic acid (siRNA) technology are also becoming important tools for transgenesis. In 2006 the European Medicines Agency (EMEA) gave green light for the commercialistion of the first recombinant protein produced in the milk of transgenic animals. Recombinant antithrombin III will be launched as ATryn for prophylactic treatment of patients with congenital antithrombin deficiency. This important milestone will boost the research activities in farm animal transgenesis. Recent developments in transgenic techniques of farm animals are discussed in this review. Additional keywords: conditional transgene expression, gene pharming, lentiviral mediated transgenesis, pronuclear DNA injection, RNA interference, somatic cloning, xenotransplantation. Introduction The first transgenic livestock were produced in 1985 via microinjection of foreign DNA into pronuclei of zygotes (Hammer et al. 1985). As microinjection has several significant shortcomings, including random integration into the host genome, low efficiency and frequent incidence of mosaicism, research has focussed on alternate methodologies for improving the generation of transgenic livestock (Kues and Niemann 2004; Table 1). These include sperm mediated DNA transfer (Gandolfi 1998; Squires 1999; Chang et al. 2002; Lavitrano et al. 2002), the intracytoplasmic injection (ICSI) of sperm heads carrying foreign DNA (Perry et al. 1999), injection or infection of oocytes or embryos by viral vectors (Haskell and Bowen 1995; Chan et al. 1998; Hofmann et al. 2003; Whitelaw et al. 2004) or the use of nuclear transfer (Schnieke et al. 1997; Cibelli et al. 1998; Baguisi et al. 1999). Further qualitative improvements may be derived from technologies that allow targeted modifications of the genome (Dai et al. 2002; Lai et al. 2002), or conditional instead of a constitutive expression of transgenes (Kues et al. 2006). Other important developments are the usage of large genomic constructs to optimise expression, the application of RNA interference to knock down specific genes and the use of episomal vectors that might allow a more consistent expression owing to the absence of position effects (Manzini et al. 2006). Pharmaceutical production in the mammary gland of transgenic animals Gene ‘pharming’ entails the production of recombinant human pharmaceutical proteins in the mammary gland of transgenic animals. This technology can overcome the limitations of current recombinant production systems for pharmaceutical proteins (Meade et al. 1999; Rudolph 1999). The mammary gland is © CSIRO 2007 the preferred production site mainly because of the quantities of protein that can be produced by employing various mammary gland-specific promoter and the ease of production and purification (Meade et al. 1999; Rudolph 1999). Numerous proteins have been produced at large amounts in the mammary gland of transgenic sheep, goat, cattle, pigs and rabbits and have been advanced to clinical trials (Kues and Niemann 2004). Phase III trials for antithrombin III (ATIII) (ATryn from GTC Biotherapeutics, Framingham, MA, USA) produced in the mammary gland of transgenic goats have been completed and the recombinant product has been approved as a drug by the European Medicines Agency (EMEA) in August 2006. The protein is expected to be on the market as a fully registered drug in 2007. Successful drug registration of ATryn demonstrated the usefulness and solidity of this approach and will accelerate registration of further products from this process, as well as stimulate research and commercial activity in this area. Other recombinant proteins from the udder are recombinant C1 inhibitor (Pharming BV, Leiden, The Netherlands) produced in the milk of transgenic rabbits, α-antitrypsin (α-AT) and tissue plasminogen activator (tPA), which have all progressed to advanced clinical trials (Kues and Niemann 2004). The enzyme α-glucosidase (Pharming BV) from the milk of transgenic rabbits has orphan drug registration and has been successfully used for the treatment of Pompe’s disease (Van den Hout et al. 2001). An overview of the current list of recombinant pharmaceutical proteins advanced state to commercial exploitation is given in Table 2 (Echelard et al. 2006). The use of somatic nuclear transfer accelerates production of transgenic animals with high mammary gland specific synthesis of recombinant proteins (Fig. 1). Another production site for recombinant products is the blood of transgenic animals.Transgenic cattle were cloned that produce 10.1071/RD07040 1031-3613/07/060762 Transgenic farm animals: an update Reproduction, Fertility and Development 763 Table 1. Milestones (live offspring) in transgenesis and somatic cloning in farm animals Modified from Niemann et al. 2005 Year Milestone Strategy Reference 1985 1986 1997 1997 1998 First transgenic sheep and pigs Embryonic cloning of sheep Cloning of sheep with somatic donor cells Transgenic sheep produced by nuclear transfer Transgenic cattle produced from fetal fibroblasts and nuclear transfer Generation of transgenic cattle by MMLV injection Gene targeting in sheep Trans-chromosomal cattle Knockout in pigs Homozygous gene knockout in pigs Transgenic pigs via lentiviral injection Conditional transgene expression in pigs (tet-off) Microinjection of DNA into one pronucleus of a zygote Nuclear transfer using embryonic cells as donor cells Nuclear transfer using adult somatic donor cells Random integration of the construct Random integration of the construct Hammer et al. 1985 Willadsen 1986 Wilmut et al. 1997 Schnieke et al. 1997 Cibelli et al. 1998 Infection of oocytes with helper viruses Gene replacement and nuclear transfer Artificial chromosome Heterozygous knockout Homozygous knock-out Gene transfer into zygotes via lentiviruses Pronuclear DNA injection and crossbreeding Chan et al. 1998 McCreath et al. 2000 Kuroiwa et al. 2002 Dai et al. 2002, Lai et al. 2002 Phelps et al. 2003 Hofmann et al. 2003 Kues et al. 2006 1998 2000 2002 2002 2003 2003 2006 Table 2. List of therapeutic proteins produced in the milk of transgenic animal that are currently in commercial development HAE, Hereditary angioedema; AFP, α-fetoprotein; HGH, human growth hormone; Mab, monoclonal antibody. Modified from Echelard et al. 2006 Products Companies Production animal Developmental stage ATryn (recombinant human antithrombin III) C1 Esterase Inhibitor MM-093 (AFP) Alpha-Glucosidase HGH Albumin Fibrinogen Collagen Lactoferrin Alpha-1 Antitrypsin Malaria vaccine CD 137 (4–1BB) MAb GTC Biotherapeutics Goat Pharming Merrimack and GTC Biotherapeutics Pharming BioSidus GTC Biotherapeutics Pharming Pharming Pharming GTC Biotherapeutics GTC Biotherapeutics GTC Biotherapeutics Rabbit Goat Rabbit Cow Cow Cow Cow Cow Goat Goat Goat EU: EMEA approved US: Phase 3 Phase 3 for HAE Phase 2 for RA Phase 2, on hold Preclinical Preclinical Preclinical Preclinical Preclinical Preclinical Preclinical Preclinical a recombinant bi-specific antibody in their blood. This antibody is stable after purification from serum and active in mediating target-cell-restricted T-cell stimulation and tumour cell killing (Grosse-Hovest et al. 2004). To ensure safety of recombinant products, guidelines developed by the Food and Drug Administration (FDA) of the USA require monitoring of the animals’ health, validation of the gene construct, characterisation of the isolated recombinant protein, as well as performance of the transgenic animals over several generations. This has been taken into account when developing the ‘gene pharming’ concept, for example by using exclusively animals from prion-disease-free countries (New Zealand) and keeping the production animals under strict hygienic conditions. Porcine-to-human xenotransplantation The progress in organ transplantation technology has led to an acute shortage of appropriate organs, and cadaveric or live organ donation can by far not meet the demand in western societies. To close the growing gap between demand and availability of appropriate organs, porcine xenografts from domesticated pigs are considered the solution of choice (Bach 1998; Platt and Lin 1998; Kues and Niemann 2004). Essential prerequisites for successful xenotransplantation are: (1) overcoming the immunological hurdles; (2) prevention of transmission of pathogens from the donor animal to the human recipient; and (3) compatibility of the donor organs with the human organ in terms of anatomy and physiology. The main strategies that have been successfully explored for a long-term suppression of the first immunological hurdle, i.e. the hyperacute rejection (HAR) of porcine xenografts, are the synthesis of human complement regulatory proteins (RCAs) in transgenic pigs (Cozzi and White 1995; Bach 1998; Platt and Lin 1998) and/or the knockout of antigenic structures on the surface of the porcine organ that cause HAR, i.e. the α-gal-epitopes (Phelps et al. 2003; Kuwaki et al. 2005; Yamada et al. 2005). Survival rates after transplantation of porcine RCA transgenic 764 Reproduction, Fertility and Development H. Niemann and W. A. Kues DNA 0.5 yr 1 yr 1.5 yr 2 yr 2.5 yr 3 yr 3.5 yr 4 yr Generate construct/cell lines Nuclear transfer F0 Female goat births F0 Induced lactation F0 Natural lactation Research material Pre-clinical material Phase 1 material F1 Females/male births F1 Females natural lactation F2 Females natural lactaton Bridging/Phase 3 material Fig. 1. Timeline for the production of recombinant proteins in the mammary gland of transgenic goats via somatic cloning (modified from Echelard et al. 2006). hearts or kidneys to immune-suppressed non-human primates ranged from 23 to 135 days. Overall, current data show that HAR can be overcome in a clinically acceptable manner by expressing human complement regulators in transgenic pigs (Bach 1998). The usefulness of porcine organs with a knockout for the 1,3-α-galactosyltransferase (α-gal) gene in a xenotransplantation situation has been demonstrated. Survival rates reached 2–6 months upon transplantation of porcine hearts or kidneys from α-gal-knockout pigs to baboons (Kuwaki et al. 2005; Yamada et al. 2005). However, donor pigs with multiple transgenes critical in other immunological barriers are thought to be required for a prolonged survival of >6 months of porcine organs in nonhuman primates. We have recently produced triple transgenic pigs to suppress the coagulatory disorder frequently found after porcine-to-primate xenotransplantation by expressing human thrombomodulin (hTM) or human heme-oxygenase-1 (hHO-1) on top of two RCAs (Petersen et al. 2006). A consistent survival of porcine xenografts for more than 6 months in non-human primate recipients is thought to be necessary for starting clinical trials with human patients. Recent research has revealed that the risk of porcine endogenous retrovirus (PERV) transmission to human patients is low, paving the way for preclinical testing of xenografts (Switzer et al. 2001; Irgang et al. 2003). RNA interference (RNAi) is a promising approach to knock down PERV expression in porcine cells (Dieckhoff et al. 2007), which could then be used in nuclear transfer. Guidelines for the clinical application of porcine xenotransplants are already available in the USA and are currently being developed in several other countries. The compatibility between porcine and human organs, remains to a large extent an enigma, but preliminary information regarding the function of porcine kidneys and heart in non-human primates are promising in this respect (Ibrahim et al. 2006). Transgenic animals in agriculture Agricultural applications of transgenic animals are lagging behind the biomedical area (Kues and Niemann 2004). Table 3 shows an overview on the various attempts to produce animals transgenic for agricultural traits. An important step towards the production of healthier pork has been made by the first pigs transgenic for a desaturase gene derived either from spinach or Caenorhabditis elegans. These pigs produced a higher ratio of polyunsaturated versus saturated fatty acids in their muscles clearly indicating the potential of rendering more healthier pork in the near future (Niemann 2004; Saeki et al. 2004; Lai et al. 2006).A human diet rich in non-saturated fatty acids is correlated with a reduced risk of stroke and coronary diseases. In the pig, it has been shown that transgenic expression of a bovine lactalbumin construct in sow milk resulted in higher lactose contents and greater milk yields, which correlated with a better survival and development of the piglets (Wheeler et al. 2001). The increased survival of piglets at weaning provides significant benefits to animal welfare and the producer. Phytase transgenic pigs have been developed to address the problem of manure-based environmental pollution. These pigs carry a bacterial phytase gene under the transcriptional control of a salivary gland specific promoter, which allows the pigs to digest plant phytate. Without the bacterial enzyme, the phytate phosphorus passes undigested into manure and pollutes the environment. With the bacterial enzyme, the fecal phosphorus output was reduced up to 75% (Golovan et al. 2001). These environmentally friendly pigs are expected to enter commercial production chains within the next few years. The physicochemical properties of milk are mainly affected by the ratio of casein variants making them a prime target for the improvement of milk composition. The bovine casein ratio can be altered by overexpression of β- and κ-casein clearly underpinning the potential for improvements in the functional properties of bovine milk (Brophy et al. 2003). Mastitis is a preeminent health problem in modern dairy cattle production causing significant economic losses. Lysostaphin has been shown to confer specific resistance against mastitis caused by Staphylococcus aureus. Cows have been cloned from transgenic donor cells that express a lysostaphin gene construct in the mammary gland, Transgenic farm animals: an update Reproduction, Fertility and Development 765 Table 3. Overview on successful transgenic livestock for agricultural production Transgenic trait Key molecule Increased growth rate, less body fat Increased growth rate, less body fat Increased level of poly-un-saturated fatty acids in pork Increased level of poly-un-saturated fatty acids in pork Phosphate metabolism Milk composition (lactose increase) Construct Gene transfer method Species Reference Growth hormone (GH) hMT-pGH Insulin-like growth factor-1 (IGF-1) mMT-hIGF-1 Desaturase (from spinach) maP2 -FAD2 Microinjection Microinjection Microinjection Pig Pig Pig Nottle et al. 1999 Pursel et al. 1999 Saeki et al. 2004 Desaturase (from C. elegans) Somatic cloning Pig Lai et al. 2006 Microinjection Microinjection Pig Pig Golovan et al. 2001 Wheeler et al. 2001 Microinjection Microinjection Microinjection CAGGS-hfat-1 Phytase α-lactalbumin Influenza resistance Enhanced disease resistance Wool growth PSP-APPA genomic bovine α-lactalbumin Mx protein mMx1-Mx IgA α,K-α,K Insulin-like growth factor-1 (IGF-1) Ker-IGF-1 Visna Virus resistance Ovine prion locus Visna virus envelope Prion protein (PrP) visna LTR-env Microinjection targeting vector Somatic cloning (homol. recomb.) Milk fat composition Milk composition (increase of whey proteins) Milk composition (increase of lactoferrin) Staph. aureus mastitis resistance Stearoyl desaturase β-casein κ-casein β-lactogl-SCD genomic CSN2 CSN-CSN-3 α-s2cas-mLF Microinjection Somatic cloning Pig Müller et al. 1992 Pig, sheep Lo et al. 1991 Sheep Damak et al. 1996a, 1996b Sheep Clements et al. 1994 Sheep Denning et al. 2001 (animals dead shortly after birth) Goat Reh et al. 2004 Cattle Brophy et al. 2003 Microinjection Cattle Platenburg et al. 1994 ovine β-lactogllysostaphin Somatic cloning Cattle Wall et al. 2005 human lactoferrin Lysostaphin rendering the animals mastitis-resistant (Wall et al. 2005). The advent to detailed genomic maps and appropriate tools for the genetic engineering of livestock species will accelerate and enhance transgenic animal production for specific production purposes. Transgenic application in other pet species, such as dog or cat, is hypothetical at present, but may become more widespread when more efficient gene transfer technologies are developed and specific genetic traits can be targeted. Some of the emerging technologies relevant in this context are described below. Transgenic pets Emerging technologies Lentiviral transgenesis Lentiviruses have been shown to be efficient vectors for the delivery of genes into oocytes and zygotes. Injection of lentiviruses into the perivitelline space of porcine zygotes resulted in a high proportion of transgenic offspring, from which expressing transgenic lines could be established (Hofmann et al. 2003; Whitelaw et al. 2004). The production of transgenic cattle via lentiviral vectors required the injection into the perivitelline space of oocytes (Hofmann et al. 2004). Lentiviral gene transfer in livestock is compatible with unprecedented high rates of transgenic animals. Whether multiple integration of lentiviral vectors into the host genome is associated with unwanted side effects, such as oncogene activation or insertional mutagenesis, remains to be investigated. However, the silencing of lentiviral sequences in transgenic lines (Hofmann et al. 2006) and the high frequency of mosaicism in founder animals requires further research. As can be seen above, transgenic approaches have focussed on livestock species either for biomedical or agricultural purposes. Recent applications of transgenic technology relate to the development of new varieties of ornamental fish. Transgenic medaka (Oryzias latipes, rice fish) with a fluorescent green colour were described in 2001 (Chou et al. 2001) and subsequently approved for sale in Taiwan. The fluorescent medaka is currently marketed by the Taiwanese company Taikong (www.azoo.com.tw). GloFish is a trademarked transgenic zebra fish (Danio rerio) expressing a red fluorescent protein from a sea anemone under the transcriptional control of a muscle-specific promoter (Gong et al. 2003). In addition, green and yellow fluorescent proteins have been introduced into stable transgenic lines, resulting in fishes with different flurorescence (Fig. 2). Yorktown Technologies (www.glofish.com) started commercial sales of the transgenic zebrafish in the USA at retail prices of approximately US$5.00. Thus, GloFish has become the first transgenic animal readily available in the USA. A recent report of the FDA revealed no evidence that Glofish represent a risk (US FDA 2003). Commercialisation of fluorescent fish has gone forward in several countries other than the USA, such as Taiwan, Malaysia, and Hong Kong, whereas marketing in Australia, Canada and the European Union is prohibited. Conditional transgenesis in farm animals Recently, we reported the first tetracycline-controlled transgene expression in a farm animal (Kues et al. 2006). An autoregulative tetracycline-responsive bicistronic expression cassette (NTA) was introduced into the pig genome via pronuclear DNA injection (Fig. 3a). The NTA cassette was designed to give 766 Reproduction, Fertility and Development H. Niemann and W. A. Kues Fig. 2. Transgenic zebrafishes expressing different fluorescent proteins. The image was taken under natural white light (reproduced with permission from www.glofish.com). Owing to high expression of the fluorescent proteins the fishes appear brightly coloured. ubiquitous expression of human RCAs independent of cellular transcription cofactors. Expression from this construct could be inhibited reversibly by feeding the animals doxycycline (tet-off system). In 10 transgenic pig lines carrying one NTA cassette, the transgene was silenced in all tissues with the exception of skeletal muscle, where the transgene was expressed in discrete muscle fibres (Niemann and Kues 2003). Interestingly, crossbreeding to produce animals with two NTA cassettes resulted in reactivation of the silenced NTA cassettes and a strong and broad tissue-independent, tetracycline-sensitive RCA expression pattern (Fig. 3b). The extent of reactivation in the double transgenic pigs correlated inversely with the methylation status of the NTA cassettes, and probably reflected effects of the different integration sites. This shows that selective crossbreeding of transgenic pig lines can overcome epigenetic silencing. This approach highlights the importance to study epigenetic (trans)-gene regulation in the pig (Fig. 3c). Artificial chromosomes as transgene vectors Artificial chromosomes have the potential to carry very large pieces of DNA that are maintained as episomal entities (Niemann and Kues 2003). A human artificial chromosome (HAC) containing the entire sequences of the human immunoglobulin heavy and light chain loci has been introduced into bovine fibroblasts, which were then used in nuclear transfer. Transchromosomal bovine offspring were obtained that expressed human immunoglobulin in their blood. This system could be a significant step forward in the production of human therapeutic polyclonal antibodies (Kuroiwa et al. 2002). Follow-up studies showed that the HAC was maintained in most animals over several years in the first generation cattle (Robl et al. 2007). Whether HACs separate correctly during meiotic cell divisions remains to be shown. Spermatogonial transgenesis Transplantation of primordial germ cells into the testes is an alternative approach to generate transgenic animals. Initial experiments in mice showed that the depletion of endogenous spermatogenonial stem cells by treatment with the chemical busulfan before transplantation is effective and compatible with re-colonisation by donor cells. Transmission of the donor haplotype to the next generation after germ-cell transplantation has been achieved in goats (Honaramooz et al. 2003). Current major obstacles of this strategy are the lack of efficient in vitro culture methods for primordial germ/prospermatogonial cells and the lack of efficient gene transfer techniques into these cells. RNA interference mediated gene knock down RNA interference is a conserved post-transcriptional gene regulatory process in most biological systems, including fungi, plants and animals. The common mechanistic elements are double stranded small interfering RNAs (siRNA) with 19–23 nucleotides, which specifically bind to complementary sequences of their target mRNAs. The target mRNAs are then degraded by endonuclease activity and no protein is translated (Plasterk 2002). RNA interference is involved in gene regulation and specifically to control/suppress the translation of mRNAs from endogenous and exogenous viral elements and can be used for therapeutic purposes (Dallas and Vlassow 2006). For a transient gene knock down, synthetic siRNAs are transfected in cells or early embryos (Clark and Whitelaw 2003; Iqbal et al. 2007). For stable gene repression the siRNA sequences must be incorporated into a gene construct. The combination of siRNA with the lentiviral vector technology is a highly effective tool in this respect. RNAi knockdown of PERV has been shown in porcine primary cells (Dieckhoff et al. 2007), as well as the knockdown of the prion protein gene (PRNP) in Transgenic farm animals: an update Reproduction, Fertility and Development (a) Inactivated by exogenous tetracycline RCA RCA Health and welfare of transgenic farm animals IRES tTA pA (b) Genotype L13 L15 1 ⫺ ⫹ 2 ⫹ ⫹ cattle embryos (Golding et al. 2006). Germline transmission of lentiviral siRNA has been shown in rats over three generations (Tenenhaus Dann et al. 2006). In contrast to knock out technologies, which require time consuming breeding strategies, RNAi can easily be integrated into existing breeding lines. Transactivator (AAA)n ptTA 767 3 ⫹ ⫺ 4 ⫹ ⫺ 5 ⫹ ⫹ 6 ⫺ ⫹ 7 ⫹ ⫹ 8 ⫹ ⫹ 9 ⫹ ⫺ 2.3 kb (c) Closed (heterochromatin) DNA demethylation DNA methylation Histone acetylation Histone deacetylation Open (euchromatin) Transcription Fig. 3. Conditional expression of transgenes in pigs and epigenetic regulation. (a) Design of an autoregulative cassette, encoding a regulator of complement activation (RCA) and a tetracycline sensitive transactivator (tTA), separated by an internal ribosomal entry sequence (IRES). Basal expression of the construct results in the translation of tTA and subsequently in autoregulative up-regulation, by binding of tTA to the promoter (ptTA ). Exogenous tetracycline binds and inactivates the transactivator (tet-off), resulting in down-regulation of transcription. (b) Reactivation of silenced transgenes by line crossbreeding. Animals of lines 13 and 15, which are hemizygous carriers for one autoregulative cassette, were crossbred to obtain double transgenic offspring. Specific detection of RCA mRNA levels by Northern blotting of muscle biopsies from nine siblings. Animals that carried one cassette, either from line 13 (L13) or line 15 (L15), expressed low levels of the transgene (lane 1, 3, 4, 6 and 9); these low expression levels were also found in the parent animals. Siblings that carried both cassette (lane 2, 5, 7 and 8) showed 50–100 times higher transcript levels. (c) Epigenetic regulation of gene expression. Simplified diagram showing that DNA regions may switch between a closed and an open confirmation. Epigenetic modification of the DNA by methylation of CG-dinucleotids and of the histones by acetylation contribute to these changes and may fix the DNA in one mode. In the open confirmation (euchromatin) the DNA is accessible to DNA binding protein, like transcription factors and RNA polymerase II, and transcription can be activated. In the closed mode (heterochromatin) a gene locus is silenced. Owing to integration and expression of transgenes, insertional mutagenesis and unwanted pathological side effects cannot be ruled out and concerns have been raised about the health of transgenic farm animals (Van Reenen et al. 2001). Transgenic farm animals have been produced since 1985 by pronuclear DNA injection, but only few systematic studies investigated the health status in cohorts of these animals. Overexpression of human growth hormone in pigs and sheep was associated with specific pathological phenotypes, which could be avoided by advanced constructs (Nottle et al. 1999). In pigs transgenic for human decay accelerating factor (hDAF), maintained at qualified pathogen free conditions, the haematology and blood chemistry were normal compared with transgenic animals (Tucker et al. 2002). With the exception of a slightly exceeded growth rate, no abnormalities were found. A detailed pathomorphological examination of nine lines of hemizygous pigs expressing human RCAs revealed no adverse effects correlating with transgene expression (Deppenmeier et al. 2006), providing clear evidence that transgenesis per se does not compromise animal health and welfare. Several of the investigated animals carried a bicistronic expression cassette, driving hCD59 and the tetracycline transactivator (Kues et al. 2006), suggesting that multi-transgenic animals are compatible with a normal health status. The hemizygous lines were fertile and produced normal litter sizes. However, attempts to breed these lines to homozygosity resulted in very small litter sizes in the first generation, likely reflecting inbreeding depression. In mice, several inbreeding rounds are necessary for extinction of a specific line (Taft et al. 2006). Cloning of mammals by somatic nuclear transfer is still tainted by low success rate of live offspring, typically with a range of 1–5% of transferred embryos surviving to term (Kues and Niemann 2004). Recently, we have obtained significant improvement of porcine cloning success by better selection and optimised treatment of the recipients, specifically by providing a 24-h asynchrony between the pre-ovulatory oviducts of the recipients and the reconstructed embryos. Presumably, this gave the embryos additional time to achieve the necessary level of nuclear reprogramming. A second major improvement was a switch to peripubertal recipients synchronised with Altrenogest feeding over 15 days (Regumate, Cilag-Janssen, Neuss, Germany, 20 mg per animal per day) rather than using the first oestrus of prepubertal gilts (Walker et al. 2002). This resulted in pregnancy rates of ∼80% and only slightly reduced litter size (Petersen et al. 2007). High embryonic and fetal attrition rates of cloned animals are specifically observed in ruminants that seem to be particularly susceptible to epigenetic aberrations. Cardiopulmonary and placental lesions and lesions in the skeletal and muscle system have been observed in cloned cattle (Hill et al. 1999). The observed adverse effects seem to be related to the cloning procedure 768 Reproduction, Fertility and Development per se and not primarily to transgenic modification, as similar adverse effects are also seen in cloned cattle without genetic modification (Panarace et al. 2006). In contrast, transgenic cloned pigs that survived the neonatal period are apparently normal in all haematologial and biochemical parameters (Carter et al. 2002). Studies with sufficient numbers of transgenic farm animals produced by emerging technologies, such as lentiviral and spermatogonial transgenesis, or expressing new constructs, like HAC and siRNA, will be necessary to assess the safety of these transgenic approaches. Outlook Recent advances in genomic technologies in farm animals present not only a major opportunity to address significant limitations in agricultural production, but also offer exciting prospects for medical research, significantly extending the important role of animals as models of human health and disease (Niemann et al. 2007). Developments in animal genomics have broadly followed the route that the human genome field has led, both in terms of completeness of data and in developing tools and applications as demonstrated by the recent availability of advanced drafts of the maps of the bovine, chicken, dog and bee genomes. The possibility of extending knowledge through deliberately engineering change in the genome is unique to animals and is currently being developed through the integration of advanced molecular tools and breeding technologies. However, the full realisation of this exciting potential is handicapped by gaps in our understanding of embryo genomics and the epigenetic changes that are critical in the production of healthy offspring. The convergence of the recent advances in reproductive technologies with the tools of molecular biology opens a new dimension for animal breeding. Major prerequisites are the continuous refinement of reproductive biotechnologies and a rapid completion and refinement of livestock genomic maps. Embryonic stem cells (ES-cells) play a critical role in this context. Despite numerous efforts, no ES-cells with germ line contribution have been established from mammals other than the mouse. ES-like cells, i.e. cells that share several parameters with true EScells, have been reported in several species. The time period over which these cells could be maintained in culture varied from 13 weeks to 3 years (Gjørret and Maddox-Hyttel 2005; Yadav et al. 2005). Derivation of true ES cells in farm animals will permit exploitation of the full power of recombinant DNA technology in animal breeding and will thus be critical to develop sustainable and diversified animal production systems. We anticipate genetically modified animals will soon play a significant role in the biomedical field. Agricultural application might be further down the track given the complexity of some of the economically important traits. Acknowledgements The financial support by a grant from the DFG (FOR 535) is gratefully acknowledged. This review contains in parts data from Niemann, H., Kues, W. A., and Carnwath, J. W. (2005). Transgenic farm animals: present and future. Rev. Sci. Tech. OIE 24, 285–298. H. 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In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Theriogenology 68S (2007) S165–S177 www.theriojournal.com Application of DNA array technology to mammalian embryos H. Niemann *, J.W. Carnwath, W. Kues Department of Biotechnology, Institute for Animal Breeding, Mariensee, D-31535 Neustadt, Germany Abstract Early embryogenesis depends on a tightly choreographed succession of gene expression patterns which define normal development. Fertilization and the first zygotic cleavage involve major changes to paternal and maternal chromatin and translation of maternal RNAs which have been sequestered in the oocyte during oogenesis. At a critical species-specific point known as the major onset of embryonic expression, there is a dramatic increase in expression from the new diploid genome. The advent of array technology has, for the first time, made possible to determine the transcriptional profile of all 20,000 mammalian genes during embryogenesis, although the small amount of mRNA in a single embryo necessitates either pooling large numbers of embryos or a global amplification procedure to give sufficient labeled RNA for analysis. Following array hybridization, various bioinformatic tools must be employed to determine the expression level for each gene, often based on multiple oligonucleotide probes and complex background estimation protocols. The grouped analysis of clusters of genes which represent specific biological pathways provides the key to understanding embryonic development, embryonic stem cell proliferation and the reprogramming of gene expression after somatic cloning. Arrays are being developed to address specific biological questions related to embryonic development including DNA methylation and microRNA expression. Array technology in its various facets is an important diagnostic tool for the early detection of developmental aberrations; for improving the safety of assisted reproduction technologies for man; and for improving the efficiency of producing cloned and/or transgenic farm animals. This review discusses current approaches and limitations of DNA microarray technology with emphasis on bovine embryos. # 2007 Elsevier Inc. All rights reserved. Keywords: Preimplantation embryo; Transcriptome; RNA amplification; DNA microarray; Bioinformatics; Plasticity of gene expression; Maternal and embryonic gene expression 1. Introduction Sequencing and annotation of the human genome resulted in a downward estimation of the total number of protein-coding genes to approximately 20,000 [1]. This is much lower than previous estimates which ranged to as many as 150,000 genes and is similar to estimates for other vertebrates’ species including mouse, chicken and pufferfish [2–6], or bovine and dog * Corresponding author. Tel.: +49 5034 871 148; fax: +49 5034 871 101. E-mail address: [email protected] (H. Niemann). 0093-691X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2007.05.041 (http://www.hgse.bcm.tmc.edu/projects/bovine [7,8]). It is clear that complex organisms exploit a variety of regulatory mechanisms to extend the functionality of their genomes [9], including differential promoter activation, alternative RNA splicing, RNA modification, RNA editing, localization, translation and stability of RNA, expression of non-coding RNA, antisense RNA and microRNAs [10–16]. It is important to note that most of these mechanisms work together at the RNA level to produce the functional transcriptome of an organism. Tissue-specific transcriptomic profiles indicate important features of the regulatory process. During the past decade, efficient high-throughput methods have been developed for whole genome sequencing, transcriptome Author's personal copy S166 H. Niemann et al. / Theriogenology 68S (2007) S165–S177 and proteome analysis [17–19]. These technologies are crucial to improve the efficiency and safety of assisted reproduction techniques, to facilitate the development of novel therapies based on regenerative medicine and to insure the quality of biotechnological products at the molecular level. The application of array technology to the analysis of preimplantation mammalian embryos poses-specific challenges associated with the picogram levels of mRNA in a single embryo, the plasticity of the embryonic transcriptome and the difficulties in obtaining in vivo developing embryos in some species. Gene expression patterns in mammalian embryos are more complex than those in most somatic cells due to the dramatic restructuring of the paternal chromatin, the major onset of embryonic transcription, events related to the cellular differentiation at the early morula stage, blastocyst formation, expansion and hatching and finally implantation. Embryo development is first dependent on maternally stored transcripts which are gradually depleted until the embryo produces its own transcripts after a switch to the embryonic expression program. The gene expression patterns and RNA stability in oocytes and early embryos prior to the activation of embryonic expression are dramatically different from what is seen after major onset of embryonic transcription. The onset of embryonic gene transcription occurs at a species-specific time point; in mice, the major activation starts in late 1-cell embryos, in pigs it occurs at 4-cell stage, in men at the 4- to 8-cell stage and in bovine embryos it is delayed until the 8- to 16-cell stage [20] (Fig. 1). Due to the late onset of this maternal-to-embryonic transition in bovine embryos, they provide an excellent model in which to study early reprogramming events in detail. Major epigenetic reorganization also occurs during preimplantation development. This includes DNA demethylation and methylation as well as targeted modifications of histone methylation and acetylation all of which play a role in controlling chromatin remodeling and X chromosome inactivation [21]. Another challenge for applying DNA arrays to preimplantation embryos is the high degree of expression plasticity seen in early stage embryos. Preimplantation stages of several species can be cultured in vitro. However, this is frequently associated with changes in chromatin configuration and gene expression [22–24]. In vitro production of bovine embryos is usually Fig. 1. Early reprogramming steps of maternal and embryonic transcriptomes are extended in bovine embryos (bottom) compared to mouse (upper) and human embryos (middle panel). Author's personal copy H. Niemann et al. / Theriogenology 68S (2007) S165–S177 necessary to provide the large numbers of embryos needed for an array analysis in this species but it is clear that the changes in embryonic transcription induced by the culture method itself can affect the transcriptional profile. With the advent of rather complete and validated genomic maps for farm animals, DNA arrays are increasingly being used to unravel the transcriptional profile during preimplantation development. Here, we discuss the current status and limitations of DNA microarray analysis as it is applied to preimplantation embryos with an emphasis of the bovine embryonic transcriptome. 2. Array analysis of preimplantation embryos 2.1. Principles of array technology The most fundamental requisite for detailed expression array analysis is availability of sufficient genomic sequences to produce an array. Because they require less hybridization solution than larger array formats, microarrays are routinely used for large scale transcriptome analyses. They have been widely and successfully employed for simultaneously monitoring the expression of complete genomes, thus identifying genes that are differentially expressed in distinct cell types, developmental stages, disease states or in cells exposed to various reagents [17]. High-density oligonucleotide arrays can be used to monitor the activity of an entire genome of a specific organism, thus defining the cellular transcriptome in the form of a snapshot of the transcription profile and revealing details of the dynamic changes. Microarrays consist of hundreds to millions of microscopically small spots attached to a matrix of glass or plastic, each containing a short but gene specific nucleic acid sequence, typically a synthetic oligonucleotide or cDNA. The microarray design can be miniaturized to few millimeters, allowing sample volumes to be reduced to a few microliters. The biological principle of the DNA array method is hybridization based on complementary base pair binding of nucleic acid molecules to their sequence matched spotted probes. A sequence of 20 bases is sufficient to identify a gene with certainty. To visualize differences in transcriptional activity, a test sample of the complex nucleic acid mixture extracted from cells or tissues is chemically tagged with a fluorochrome or chemical conjugate and then hybridized to the array. This results in a signal at each gene specific spot whose intensity is due to retention of some amount of its complementary labeled nucleic acid. The hybridization signals are quantitatively recorded and the amount of complementary nucleic acid at each point is calculated S167 with respect to local background. In some cases, this involves averaging the results from several independent spots all representing different locations within the target gene. The number of genes from which expression can be simultaneously detected, is potentially unlimited and whole genome arrays are commercially available. In principle, genome coverage on an array is only limited by the density and number of spots on the array. Array analysis depends first and foremost on the determination of probability that a transcript is present or absent. This is based on statistical analysis of the strength of the signal above background often using several probes for the gene and often using several repeated array hybridizations. There are a variety of different statistical methods which can be used for this step and the choice of which to use depends on the goals of the experiment. One must decide between having more false positives or false negatives. The strength of the statistical analysis is increased by the number of repeated hybridizations but can also be increased by grouping genes into clusters or pathways which can be used to smooth out individual variation. Analysis of the vast amount of data requires sophisticated bioinformatic tools [25]. The objective of a microarray experiment is usually to improve the understanding of biological mechanisms. This requires bioinformatic methods which group the detection profiles of individual genes based on current knowledge of signaling pathways or gene function. Several databases, such as Gene Ontology (GO; http://www.geneontology.org) and KEGG, the Kyoto Encyclopaedia of Genes and Genomes, (http://www.genome.jp/kegg) are available for this and can readily be accessed over the internet. Further bioinformatic tools and a variety of statistical methods have been developed to refine reliability. It is usually necessary to repeat each array analysis 2–3 times to achieve satisfactorily accurate measurements. The most important criteria for evaluating any microarray analysis are reproducibility of the data, specificity of target gene detection, local background and gene specific background. Certain standards have been agreed upon and strict adherence to criteria described as Minimal Information about a Microarray Experiment (MIAME) is absolutely essential to guarantee consistent and reproducible datasets for the scientific community [26]. 2.2. Technical considerations A specific problem encountered in the array analysis of embryonic gene expression is known as the loading artifact. The necessity of loading a standardized amount Author's personal copy S168 H. Niemann et al. / Theriogenology 68S (2007) S165–S177 of labeled RNA onto each array for hybridization causes a distortion of the quantitative results because of the large change in the amount and composition of mRNA which occurs throughout preimplantation development but particularly at the major onset of embryonic gene expression. The blastocyst contains approximately twice as much mRNA as the oocyte mainly due to the greater number of individual mRNAs expressed in the blastocyst. When the total amount of labeled RNA applied to the oocyte array is the same as that applied to the blastocyst array, the amount of probe for each individual transcript in the blastocyst array is halved. Blastocyst transcripts which are close to the detection limit are lost and what appears to be a 50% reduction in transcription between the oocyte and the blastocyst for an individual gene may be only due to the loading artifact. If it would be possible to establish a group of genes whose expression levels were absolutely constant throughout preimplantation development, one could correct for the loading artifact. Similarly, if it would be possible to accurately measure the total amount of mRNA at each developmental stage, this information could be used to correct for the loading artifact. However, RT-PCR analysis has not yet revealed any gene whose expression is held at a constant level over preimplantation development, making accurate measurements of the mRNA in embryos under various culture conditions problematic. In interpretation of array results, one must realize that a number of genes, transcribed at low level in the blastocyst, are not detected because of the loading artifact and there is a systematic underestimation of the number of new genes expressed during development. One of the most commonly used methods for mRNA purification from embryos is based on poly-dT coated magnetic beads. These hybridize with the poly-A tail of mRNA and pull the mRNA out of the solution. It is important to realize that the length of the poly-A tail has an influence on the extraction efficiency. Poly-A tail length changes during embryonic development, especially between the oocyte stage where mRNA is protected and stored for later use and subsequent stages where mRNAs are transcribed for immediate use. The XIST gene provides an interesting biological example. This gives transcripts of several different lengths, which are distinguished by variations at the 30 end. Some of these have a signal for polyadenylation and some do not [27]. Thus, extraction with poly-dT coated magnetic beads will distortion the picture of transcription from this gene. Poly-dT extraction results in a second artifact due to the fact that 50 fragments of mRNA molecules which have no poly-A sequence will be lost while 30 fragments will be retained. The probability of fragmentation is a function of the length of the transcript. One routine way to detect this artifact, which is a reflection of RNA quality, is to compare spots matching 30 sequences of selected genes with the signals from 50 spots for the same genes. An array where this distortion is noted should not be included in the analysis. A commonly used RNA amplification method for Affymetrix array hybridization is the ‘‘Whole Transcript (WT) Sense Target Labeling Assay’’ (www.affymetrix.com). The system most suitable for preimplantation embryo analysis is the ‘‘Two-Cycle Target Labeling and Control Reagent package’’ which includes a protocol for target preparation from 10 to 100 ng of total RNA (see www.affymetrix.com). One of the most difficult specimens to obtain for embryonic expression analysis is a naturally developing embryo. Even embryos flushed directly from the oviduct may have altered gene expression patterns due to the nutritional status of the donor, the use of superovulation, exposure to flushing medium such as PBS, exposure to non-physiological levels of oxygen and temperature variations. There is no question that in vitro culture affects gene expression [22–24]. For example, RT-PCR analysis shows that the expression of heat shock protein increases continuously during embryonic development in vitro. The time of exposure to non-physiological conditions between flushing and freezing will influence relative expression data as it is known that different transcripts have different halflives. As a general rule, long transcripts are rare and have shorter half-lives. Experiments with embryos must be designed to control for such artifacts by minimizing the time between isolation and cryopreservation and must be interpreted with full knowledge of their limitations. The information which can be obtained by array analysis can be either the timing of (detectable) gene expression, the relative amount of individual gene expression between matched samples, the number of different genes detected in different samples using the same array and complex generalizations with respect to clustered genes and pathways. When artifacts have been minimized, the paired sample approach is a powerful method for analyzing expression. The number of arrays used in a specific analysis may be limited by practical or financial constraints but current analytical programs are quite capable of integrating the results from hundreds of arrays. The sensitivity of analysis can generally be enhanced by increasing the number of arrays used in an experiment but even when the number of arrays is restricted, it is possible to increase the sensitivity of Author's personal copy H. Niemann et al. / Theriogenology 68S (2007) S165–S177 analysis and the information content of the study by performing multivariate statistical analysis of, for example, clusters of genes related to the same pathway. This has the advantage of averaging out the fluctuations of weak signals. Array production is consistent, so that the comparison of results from two or more individual arrays is both possible and recommended. In some systems, additional sensitivity can be obtained by applying two differentially labeled samples to a single array. One of the most robust measurements is determination of the subset of strongest expressed genes at any stage since this avoids problems of sensitivity, RNA stability and statistical fluctuation. The main limitation for this form of analysis is saturation of individual spots on the array which will affect the relative rankings of highly expressed genes. Interpretations of biological effects seen in array analysis must be confirmed by RT-PCR verification. 2.3. Global amplification strategies Early embryogenesis requires the well orchestrated expression of genes to ensure normal development, and is associated with significant physical reorganization of the epi-genome and of the transcriptome. It is estimated that 5000–10,000 genes are simultaneously expressed at carefully controlled levels. Transcripts for key transcription factors may be present in only a small number of copies, while transcripts encoding major structural proteins, such as MSY2/fbx2 can represent 2% of the mRNA pool [28]. Estimations of the RNA content of mammalian oocytes range from 0.2 to 2.4 ng and blastocysts are estimated to have roughly twice as much, 0.4–5 ng [29,30]; approximately 10–20% of the total RNA consists of polyadenylated mRNA. Protocols for array hybridization typically require as much as 5–10 mg of purified nucleic acids (www.affymetrix.com). Thus samples with tiny amounts of RNA, such as laser dissected biopsies or early embryos, require unbiased global amplification prior to array analysis. The amount of mRNA, which can be isolated from a single embryo, needs to be amplified for 10,000 times to obtain sufficient material for one hybridization reaction. It is critical that this amplification does not distortion the relative proportions of mRNA found in the original mRNA pool. During the past few years several methods have been published that are compatible with efficient amplification of RNA isolated from single cells, embryos or small pools of embryos without distortion of the RNA pool ([30–33], S169 http://www.affymetrix.com/, http://www.epicentre.com, http://www.genisphere.com). Crucial steps in these global amplification protocols are the conversion of mRNA into cDNA by reverse transcriptase and second strand cDNA synthesis, followed by amplification and labeling by in vitro transcription (IVT) using T7 RNA polymerase. To introduce the recognition sequence for T7 RNA polymerase, synthetic oligo(dT) primers which include a T7 recognition sequence are used for the reverse transcription reaction. Some protocols combine the T7 RNA polymerase reaction with the polymerase chain reaction (PCR) to achieve the necessary amplification [30,31]. Affymetrix, one of the prominent companies in array analysis, recommends two cycles of IVT by T7 RNA polymerase. For any protocol, a rigorous validation of the amplification is essential to obtain meaningful hybridization data (Fig. 2). Less amplification is generally better as there will always be differences in efficiency due to variations in the length and nucleotide composition of the mRNA species. Semi-quantitative RT-PCR or Real Time PCR assays comparing amplified and non-amplified material with a broad panel of transcripts are important for validation of the amplification protocol. 2.4. The value of cross-species hybridization The rapid improvements of cDNA-chip technologies and the availability of multi-species sequence databases, e.g. GO and KEGG, have made it possible to compare gene expression profiles both within a single organism and across a wide variety of organisms on a large-scale. Cross-species gene-expression comparison is a powerful tool for the discovery of evolutionarily conserved mechanisms and pathways of expression control. The advantage of cDNA microarrays is that broad areas of homology are compared and hybridization probes are sufficiently large that small inter-species differences in nucleotide sequence do not significantly affect the analytical results. This comparative genomic approach allows one to evaluate a common set of genes within a specific developmental, metabolic, or diseaserelated gene pathway in various experimental systems. Using microarrays in studies of mammalian species other than man and rodents, e.g. non-human primates, cattle, sheep and pigs, should advance our understanding of human health and disease. This is already evident in the field of drug target validation. The lack of adequate sequence data [7,34] and commercial microarrays has limited the analysis of economically and scientifically important large domestic species such as cattle, pigs and sheep. Author's personal copy S170 H. Niemann et al. / Theriogenology 68S (2007) S165–S177 Fig. 2. Reproducibility of a cDNA array analysis with RNA isolated from single mouse embryos. The correlation coefficient of 0.98 indicates that the array provides reproducible results (for details see Brambrink et al. [31]). This is rapidly changing and cross-species hybridization experiments based on human sequence based arrays have been shown to be suitable for comparative genome expression studies. For example, array analyses have been performed using monkey, pig, mouse and salmon RNA on human oligonucleotide arrays [35–39]. An extremely important aspect of cross-species hybridization is data reproducibility. A poorly conceived cross-species hybridization experiment leads to high variability. Statistical variation can be overcome by increasing the number of samples (biological and technical replicates) and the arrays themselves can be improved by careful attention to the use of conserved (homologous) regions. We have investigated the use of cross-species hybridization for orthologous genes within a defined developmental and metabolic pathway using human and bovine fetal brain RNA as paired Cy-dye labeled targets on single human cDNA microarrays [40]. The microarrays included probes for 349 genes; each spotted 20 times to give robust statistical analysis. Validation of the results was accomplished by the use of semi-quantitative RT-PCR. Other approaches to validation include Real Time PCR and Northern blot analysis. Employing high stringency hybridization and washing, followed by analysis of the array data with appropriate statistical tests, revealed only slight interspecies differences in the expression patterns and high reproducibility of the signals for many genes. The correlation coefficient of cross hybridization between orthologous genes was 0.94. Verification of the array data by semi-quantitative RT-PCR using common primer sequences permitted coamplification of both human and bovine transcripts [40]. Results of the study demonstrated the power of comparative genomics and the feasibility of using human microarrays for the identification of differentially expressed genes in other species. 3. Current status of embryo transcriptomics 3.1. Embryos from farm animals Farm animal breeders suffer major economic losses through high early embryonic mortality, in cattle 35–50% of the fertilized oocytes fail to develop [41]; this figure is dramatically increased in modern short-interval breeding regimens [22]. In many parts of the world, large numbers of embryos are routinely produced using in vitro fertilization of oocytes collected by non-surgical ultrasound-guided follicular aspiration or harvested from slaughterhouse ovaries. Cloning by somatic cell nuclear transfer is also well established for this species reflecting, at least in part, the enormous commercial interest in improved bovine embryo technology. The abundant availability of such material provides researchers with excellent opportunities to study the causes of embryonic loss in bovine and other species, including man which show significant similarities in preimplantation development. Specific similarities include the time course of preimplantation development; switch from Author's personal copy H. Niemann et al. / Theriogenology 68S (2007) S165–S177 maternal to embryonic genomic activity, metabolism and certain pathologies associated with in vitro produced embryos. Studies using sensitive Reverse Transcription–Polymerase Chain Reaction (RT-PCR) revealed approximately 200–250 genes that were differentially expressed in in vitro derived embryos as compared to embryos flushed from the uterus [24]. Using this technology, we also identified expression anomalies in blastocysts produced from prepubertal oocytes which appear to correlate with their reduced developmental potential [42]. Oocytes from prepubertal animals are of interest because they are the source of embryos in breeding programs which are based on shortened generation intervals. The sequencing of cDNAs, e.g. expressed sequence tags (EST), from genes specifically expressed during early development is important for in-depth studies on the molecular mechanisms underlying mammalian preimplantation development. This work is well advanced in mice where >140,000 ESTs from all developmental stages are available from public databases, however, in other species, a more limited amount of sequence information is available [43]. The main limitation for embryo transcriptomics is that the establishment of a conventional cDNA EST library requires roughly 5000 oocytes, 13,500 2-cell stages, 2800 8-cell embryos or 600 blastocysts [44], which is difficult for a uniparous species. The advent of cDNA array technology has for the first time made possible to compare the global transcription pattern of 20,000 genes during mammalian embryogenesis limited only by the availability of cloned cDNA from these early stages. Application of cDNA array technology has been reported for human, mouse, bovine and porcine preimplantation embryos. A study of the transcriptional profile of 2-cell mouse embryos included 4000 genes from a cDNA-library and revealed many previously unknown transcripts, including mobile genetic elements that appear to be associated with activation of the embryonic genome [45]. The mRNA expression profile of porcine oocytes, 4- and 8-cell stages and blastocysts was studied with the aid of a 15 K cDNA-array specific for porcine reproductive organs. The transcription profile of in vivo developing embryos was compared with that of in vitro produced 4-cell, 8-cell and blastocyst stage embryos. This revealed 1409 and 1696 differentially expressed (in vivo/in vitro) genes at the 4- and 8-cell stage, respectively [46]. Studies of bovine embryonic mRNA expression based on subtractive hybridization and cDNA-arrays S171 have identified differentially expressed genes between bovine oocytes derived from large or small follicles. A total of 21 upregulated genes were found out of 1000 clones [47]. Using a similar subtraction hybridization protocol, the sequencing of 185 clones revealed 146 non-redundant genes which were associated with bovine oocyte maturation [48]. Cross-species hybridization of a human cDNAArray with bovine RNA, derived from rather large pools of >200 oocytes yielded a first expression profile for bovine oocytes before and after maturation. The relative amount of mRNA remained constant during in vitromaturation. Approximately 300 genes could positively be identified [49]. Recently, we have investigated the mRNA expression profiles of bovine oocytes and blastocysts by using a cross-species hybridization approach employing an array consisting of 15,529 human cDNAs (The ENSEMBL chip) as probe, thus allowing identifying conserved genes during human and bovine preimplantation development [50]. The analysis revealed 419 genes that were expressed in both, oocytes and blastocysts. The expression of 1324 genes was detected exclusively in the blastocyst, in contrast to 164 in the oocyte, including a significant number of novel genes. Typically genes indicative for transcriptional and translational control such as ELAVL4, TACC3 were over-expressed in the oocyte, whereas genes indicative for numerous pathways were over-expressed in blastocysts. Transcripts implicated in chromatin remodeling were found in both oocytes (NASP SMARCA2, DNMT1) and blastocysts (H2AFY, HDAC7A). Pathway analysis revealed differential expression of genes comprising 107 distinct signaling and metabolic pathways. Expression patterns in bovine and human blastocysts were to a large extent identical [50]. A major advantage of our cross-species hybridization approach is that developmentally conserved novel and annotated marker genes could be identified for human and bovine oocytes and blastocysts. Analysis with a self constructed bovine array containing 2000 clones with ESTs from different embryonic developmental stages (Blue Chip) provided insight into the expression profile during bovine embryonic development [51]. Using this Blue Chip, it was possible to link a specific embryonic transcriptional profile, determined from embryo biopsies with the outcome of embryo transfers to recipients. Embryos leading to pregnancies specifically over-expressed genes needed for implantation and placenta formation, carbohydrate metabolism, and growth factor signaling [52]. Combining subtractive hybridization with this bovine cDNA-array allowed the identification of Author's personal copy S172 H. Niemann et al. / Theriogenology 68S (2007) S165–S177 previously unknown bovine oocyte specific genes. From a total of 850 clones, 491 known genes were identified in bovine oocytes, plus 188 previously non-characterized sequences, 105 unknown sequences and 86 noninformative sequences [53]. Among the 450 clones derived from bovine blastocysts, 301 known genes, 10 unknown sequences, and 139 non-informative sequences were positively identified [51]. Other laboratories have also designed home made arrays based on either oligonucleotide or cDNA probes [54,55], which cover only a portion of bovine genome. These data illustrate the enormous challenges to arrive at a complete transcriptomic map for bovine preimplantation development. Affymetrix commercially distributes a bovine genomic array covering probe sets of approximately 23,000 transcripts and 19,000 UniGene clusters, which is based on the bovine genome sequence data. Recently, the transcriptomes of bovine in vitro matured oocytes and in vitro produced 8-cell stages were analyzed by this Affymetrix bovine array and genes critically involved in the major activation of the embryonic genome were determined [56]. Approximately 370 genes with functions in chromatin modification, apoptosis, signal transduction, metabolism and immune response were differentially expressed between oocytes and 8-cell stages, including DNMT1, DNMT2 and MeCP2 that were specifically upregulated in 8-cell stages. Large scale transcriptomic analyses are useful for unraveling the consequences of in vitro production techniques such as in vitro fertilization and culture and somatic cloning by comparison with in vivo derived embryos. Interestingly, the transcriptomes of cloned bovine embryos were found to be more similar to that of in vivo blastocysts than to in vitro produced embryos [57], suggesting that the cloned embryos successfully underwent major reprogramming. It is well documented that the in vitro conditions induce alterations in embryonic transcript profiles [22,23]. Recently, we have hybridized RNA from in vivo produced bovine oocytes and all stages of bovine preimplantation development until the blastocyst stage on the bovine Affymetrix array. For the first time this analysis will yield a complete picture of mRNA expression in the processes of oocyte maturation, fertilization, embryonic activation, and cell lineage differentiation in a large mammalian species (unpublished data). The power of cDNA array technology for unraveling critical reproductive pathways has also been demonstrated [58–60]. The developmental potential of bovine oocytes was correlated with the level of follistatin transcripts [60]. Transcript levels of genes involved in transcription and translation were linked with the origin of embryos, i.e. either in vivo fertilization or in vitro production [59]. Another study showed that transcript abundances for follicle stimulating hormone receptor, estrogen receptor 2, inhibin alpha, activin A receptor type I, cyclin D2 and two pro-apoptotic factors decreased in granulosa cells during the growth of the dominant follicle [58]. 3.2. Embryos from mice and men Extensive transcriptomic analyses have been made in mouse oocytes and preimplantation embryos [31,33,61–64]. Surplus human oocytes or embryos discarded from IVF procedures have been used in most transcriptomic studies [65–69]. Recently, RNA was isolated from pools of human metaphase II oocytes obtained after hormonal ovarian stimulation [30]. The globally amplified transcripts were then hybridized to a human genome chip covering 47,000 transcripts. A comparison of the hybridization signals with those obtained for several somatic tissues revealed that 5331 transcripts were up-regulated and 7074 transcripts were down-regulated in oocytes. Compared with data from mouse oocytes a common set of 1587 transcripts was found, which may indicate genes with conserved function in mammalian oocytes. Common sets of transcripts were also found in the transcriptomes of human oocytes and human and murine embryonic stem cells. Most prominent were transcripts encoding proteins active in the transforming growth factor (TGF)-b signaling pathway. A comprehensive genome-wide study of gene transcripts has been performed on 12 stages of murine in vivo oocytes at the germinal vesicle, and metaphase II stage and preimplantation embryos [33]. More than 4000 genes were differentially expressed during preimplantation development. Two major groups of gene activity could be defined, one comprising oocytes and first cleavage stages, and the other consisting of 4cell to blastocyst stages, thus correlating with the transition of maternal to zygotic expression. Several transcripts of molecules involved in Wnt, bone morphogenetic protein (BMP) or Notch signaling pathways were identified in the embryonic transcriptome, indicating that these critical regulators of cell fate and patterning are conserved and functional in these stages of preimplantation development [61–63]. A gene atlas of the protein-coding transcriptomes of mouse and man has been recently described for several somatic tissues [70]. On average, 8000 transcripts Author's personal copy H. Niemann et al. / Theriogenology 68S (2007) S165–S177 were determined for individual mouse tissues; 1–3% of the analyzed transcripts were detected in all analyzed tissues, i.e. 79 man and 61 mice, respectively. The abundance of these ubiquitously expressed transcripts (housekeeping genes) was found to be significantly higher than that for all tissue specific genes. 4. Limitations and perspectives of DNA arrays 4.1. Limitations A major limitation of array technologies is our fragmentary understanding of genome organization and function. Recent data suggest that various mechanisms, including epigenetics, microRNAs, non-coding and unconventional RNAs significantly contribute to the dynamics of the transcriptome [10–16]. Even the most recent arrays with densities of >1 million targets per array do not cover this complexity. The transcriptome of somatic cells seems to be regulated in a more stochastic manner than previously thought [71]. Furthermore, the enormous plasticity and highly dynamic nature of early embryonic development may hamper attempts to obtain a true representative picture of the transcriptome. It has to be born in mind that current technologies only provide snapshots of parts of the entire embryonic transcriptome which may then be further exploited by arrays specific for certain pathways and/or differentiation status, etc. Targeted approaches to screen for novel transcripts via expressed sequence tag libraries may contribute to a more complete inventory of the transcriptome [45,72]. However, even in light of these limitations, the currently available technology allows discovery of novel genetic information. Appropriate statistical methods have been developed for controlling the quality of microarrays, to ensure the reproducibility of results and to identify differential expression [73,74]. Bioinformatic methods have been adapted and refined in the past few years and now take into account inherent limitations of the array technology. A recent comprehensive comparison across various studies indicated high correlation even if different platforms and bioinformatic tools had been employed [75], thereby confirming the validity of the microarray concept across different laboratories. 4.2. Perspectives DNA array technology is based on the specific detection of sequences present in certain genes. Tiling and exon arrays will extend this technology by the comprehensive identification of individual exons [76] or S173 other DNA elements. The human Exon 1.0 ST array (Affymetrix) is spotted with targets of approximately 1 million known and predicted exons. Exon arrays offer a fine-tuned picture of gene expression, in view of the fact that the majority of mammalian genes is differentially spliced. In extreme cases, such as the neurexin gene family more than 1000 splice variants of three genes are known [77,78]. Recently, a close correlation between array data from exon and conventional gene arrays has been determined in a cell culture model [79]. This supports the concept of developing useful exon arrays. In the context of embryo transcriptomics, exon arrays will be important to reveal splice variants of genes critical in early development. Tiling arrays carry genomic DNA sequences; ideally they cover the whole genome of an organism. They can be used to study single nucleotide polymorphisms (SNP) or copy number variations (CNV), that are deletions, insertions and duplications from 1 kilobasepairs to several megabasepairs. Recent studies with tiling arrays investigated the CNV in the human genome and provided compelling evidence for an unexpected high frequency of CNVs [80]. Up to 3000 genes were found to be associated with CNV. The occurrence of this phenomenon in mice and ape suggests that a similar situation may exist in farm animals, which remains to be shown. Epigenetic regulation by DNA methylation is a widespread phenomenon in biology and bears substantial information with regard to cell physiology, dysfunction and differentiation. Primary targets of methylation are cytosine guanine dinucleotides (CpG); cytosine is modified to become 5 methyl cytosine. In the mammalian genome CpG sites are unevenly distributed and mostly clustered as CpG islands, which are often associated with promoter elements [81]. Cytosine methylation is a conserved epigenetic mechanism involved in silencing of transposons, genomic imprinting and regulation of gene expression. DNA methylation is critically important for normal development of mammals, as demonstrated by embryonic lethality of knock-outs of murine methyltransferase genes DNMT1 or DNMT3a/b. Aberrant methylation patterns are associated with developmental anomalies and specific diseases, including cancer [82,83]. Methylation sensitive arrays can detect cytosine methylation of genomic DNA and may thereby provide information on the underlying mechanisms of a specific transcriptional profile. A genome wide identification and functional analysis of methylated DNA nucleotides will provide cues on how and to which extent methylation and gene expression are connected. Author's personal copy S174 H. Niemann et al. / Theriogenology 68S (2007) S165–S177 Recently the first genome-wide DNA methylation map has been obtained in the plant Arabidopsis [84]. Methylated and non-methylated genomic DNAs were fractionated by immuno-precipitation with a monoclonal antibody that specifically recognizes methylcytosine, followed by amplification and hybridization to whole genome arrays. Pericentromeric regions, repetitive sequences, and regions coding for small interfering RNA were found to be heavily methylated. Several constitutively expressed genes showed methylation within the transcribed regions, but only rarely in promoter regions, indicating that methylation marks have different functions in different gene compartments. Employing a similar strategy, a DNA methylation profile has been obtained for individual human chromosomes [85], and the DNA methylation pattern of the tumor suppressor gene p16INK4A has been determined [86]. Several human health related technologies rely on the utilization of non-human primates and farm animals for vaccine development, organ transplantation, production of recombinant proteins and viral pathogenesis [87]. The application of animal products as drugs or treatments of human patients requires the absence of any unwanted contaminations. DNA microarrays provide a mean for a high throughput testing of these products, including products of transgenic animals [88]. Ribonucleic acid interference (RNAi) allows detailed study of biological mechanisms underlying normal and aberrant embryonic development. RNAi is a conserved post-transcriptional gene regulatory process found in most biological systems. The common mechanistic elements are small interfering RNA (siRNAs) with 21–23 nucleotides, which specifically bind complementary sequences of their target mRNAs and shut down expression. The target mRNAs are degraded and no protein is translated [89]. The specificity of the RNAi approach is shown by reduced levels of the target transcript and unchanged levels of housekeeping mRNAs [90]. Microarray analysis offers the possibility to obtain a complete profile after RNAi application on the downstream effects as well off target effects in a comprehensive way. Array based analysis forms the basis for systematic characterization of the biological function of genes in preimplantation development by high-throughput RNAi screening. Genome wide screening in Caenorhabditis elegans and Drosophila, and to a lesser extent in mammalian cells, has proven to be a valuable tool for the dissection of biological pathways. Recent developments include strategies to combine genome-scale RNAi libraries with high-throughput screening technologies and integrated high-content bioinformatic analysis [91]. Similarly, analysis of the vast database of array data obtained from cancer studies will exploit the similarities between tumor cells and embryonic cells and will increase understanding of the pathways involved in embryonic development. For example, studies in zebrafish show that embryonic and tumorigenic pathways converge via Nodal signaling [92]. The bovine embryo with its advanced in vitro techniques and established technology for recovery of in vivo embryos will be useful tool in this context. Cross-species transcriptome analyses will be instrumental to identifying evolutionary conserved pathways which are crucial for development. Alteration of specific pathways via RNAi or transgenesis will reveal physiological relationships and the genetic factors involved in aberrant development. 5. Conclusions Broad determination of the embryonic transcriptome at each developmental stage through array technology is critical for understanding the critical regulatory pathways in early development. This is leading to the discovery of new regulatory mechanisms and is identifying new roles for genes known in other contexts. At present, our understanding of the results of array analysis is limited by our fragmentary understanding of pathways involved in the regulation of chromatin structure and the pathways involved in the regulation of the transcription of individual genes. Recent developments, including exon gene arrays, microRNA arrays and methylation sensitive arrays address some of these challenges. Furthermore, it should be taken into account that ultimately the protein determines the function of a specific gene, and future studies will unravel the proteome of an embryo by appropriate protein chips. In the context of mammalian embryonic development, it is crucial to employ these new tools to obtain a complete picture of the dynamic embryo transcriptome. Array technology in its various facets is an important diagnostic tool for early detection of developmental aberrations and for improving the safety of new reproductive technologies such as transgenesis and somatic nuclear transfer [87]. Acknowledgement The authors gratefully acknowledge the financial support from DFG for the embryonic transcriptomic studies cited in this review (Ni 256/27-1, FOR 478). Author's personal copy H. 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CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources 2008 3, No. 036 Review Reproductive biotechnology in farm animals goes genomics Wilfried A. Kues, Detlef Rath and Heiner Niemann* Address: Department of Biotechnology, Institute of Farm Animal Genetics (FLI), Mariensee, Höltystrasse 10, D-31535 Neustadt, Germany. *Correspondence: Heiner Niemann. Fax: +49 (0)5034-871-101. Email: heiner.niemann@fli.bund.de Received: Accepted: 4 February 2008 23 May 2008 doi: 10.1079/PAVSNNR20083036 The electronic version of this article is the definitive one. It is located here: http://www.cababstractsplus.org/cabreviews g CAB International 2008 (Online ISSN 1749-8848) Abstract The breeding of domestic animals has a longstanding and successful history, starting with domestication several thousand years ago. Modern animal breeding strategies, predominantly based on population genetics, artificial insemination (AI) and embryo transfer (ET) technologies led to significant increases in the performances in domestic animals and are the basis for the regular supply with high-quality animal-derived food at acceptable prices. A recent addition to this arsenal of technologies is the separation of spermatozoa into X- and Y-chromosomes bearing populations by means of flow cytometry, which is going to be transferred into commercial cattle breeding. Moreover, reproductive biotechnologies are currently being complemented by new measures of molecular genetics. The genomes of several domestic animals have been sequenced and annotated. Important uses of genomic data include improving selective breeding strategies, studying expression patterns (transcriptomics or proteomics) and the production of transgenic animals tailored for specific production purposes. The technology for the production of transgenic animals, either by additive gene transfer or by selective deletion (knockout), has been advanced to the level of practical application. A crucial step in this context was the development of somatic cell nuclear transfer (SCNT) cloning, which has replaced the previously used rather inefficient method of microinjection for producing transgenic animals. In this review, we discuss recent advances in important areas of animal biotechnology related to agricultural applications, including sperm sexing, somatic cloning, genome and transcriptome research, transgenesis and recombinant DNA vaccines with emphasis on large domestic species. Keywords: Domestication, Reproductive techniques, Genome sequencing, Sperm sexing, Recombinant DNA, Somatic cloning, Embryo transcriptomics, Transgenic livestock, Agriculture Introduction: Evolution of Animal Breeding and Biotechnology The breeding of domestic animals has a longstanding and successful history, starting with domestication several thousand years ago, by which Man kept animals in his proximity and used products thereof [1]. Using the technical options that were available in each time period, humans have propagated those populations that were deemed useful for their respective needs and purposes. Selection mostly occurred according to the phenotype and/or because of specific traits. Scientifically based animal breeding has only existed for 50 years, mainly based on population genetics and statistics. A vast number of phenotypically different breeds with desirable traits resulted from this process in a short time period (in evolutionary terms). A good example is cattle, for which nowadays >800 cattle breeds can be found worldwide [2]. These breeds are not just variations of the same archetype, but differ in qualitative and quantitative characteristics, including specific disease resistance, climate adaptation, lactation performance, fertility, meat quality and nutrient requirements [3]. Modern animal-breeding strategies are based on a number of biotechnological procedures, of which artificial insemination (AI) is the most prominent example. Today, http://www.cababstractsplus.org/cabreviews 2 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources AI is employed in more than 90% of all sexually mature female dairy cattle in countries with advanced breeding programs [4]. Application of AI is also steadily increasing in pigs, where now on a global scale approximately 50% of sexually matured sows are fertilized by AI. AI allows effective propagation of the genetic potential of valuable sires within a population. On an average, 200–500 insemination doses can be produced from one bull ejaculate, which can be successfully stored frozen; the corresponding figure for the boar is 10–35 [4]. A more recent development is the separation of spermatozoa into X- and Y-chromosomes bearing populations to allow the use of semen carrying predetermined sex chromosomes [5]. In the second half of the 1980s, embryo transfer (ET) technology has been introduced into animal breeding. ET allowed for the first time a better exploitation of the genetic potential of female animals. On an average, 5–10 viable embryos are being collected from a superovulated donor cow and approximately 600 000 in vivo collected embryos are transferred worldwide, of which 50% are used after freezing and thawing; in addition, a significant number of the transferred embryos (300 000) are derived from in vitro production (in vitro maturation, fertilization and culture of embryos), i.e. mainly from abattoir ovaries [6]. In contrast to AI, ET is predominantly used in the top 1% of a breeding population. These breeding strategies, predominantly based on population genetics, AI and ET technologies led to significant increases in the performances in domestic animals and are the basis for the regular supply with high-quality animal-derived food at acceptable prices. However, three major disadvantages have to be taken into account: (1) the genetic progress is slow, at only 1–3% per year; (2) it is not possible to separate the desired traits from undesired ones; and (3) a targeted transfer of genetic information between different species is not possible. This situation is about to change. Reproductive biotechnologies are currently being complemented by new measures of molecular genetics. The genomes of several domestic animals have been sequenced and annotated, including cattle, chicken, horse, bee and dog, and the pig genome is expected to be drafted shortly (www. ensembl.org). The technology for the production of transgenic farm animals, either by additive gene transfer or by selective deletion (knockout), has been advanced to the level of practical application ([7–11]; reviewed in [12]). A crucial step in this context was the development of somatic cell nuclear transfer (SCNT) cloning [13], which has replaced the previously used rather inefficient method of microinjection for producing transgenic animals [14]. Transgenic animals have been advanced to practical application in biomedicine, in particular for the production of pharmaceutical proteins in milk (gene pharming: GTC Biotherapeutics, Pharming, Biosidus etc.) and xenotransplantation, i.e. the production of porcine organs and tissue in human organ transplantation to cope with the shortage of human organs in terminally ill patients. Agricultural applications are now rapidly emerging. Here, we discuss recent advances in important areas of animal biotechnology related to agricultural applications, including sperm sexing, somatic cloning, genome and transcriptome research, transgenesis and recombinant DNA vaccines with emphasis on large domestic species. Sperm Sexing The reliable control of the sex ratio permits faster genetic progress, higher productivity, improves animal welfare by reducing the incidence of dystocia in cattle, avoiding castration of male pigs, and producing less environmental impact through elimination of animals with the unwanted sex. In mammals, the sex-determining region (SRY) has been mapped to the short arm of the Y-chromosome [15]. SRY induces the formation of the primary testicular tissue in the genital ridge, with the further development to male reproductive organs being primarily under hormonal control. Sperm from mammalian livestock species show a difference in the relative amount of DNA between X- and Y-chromosomes bearing sperm in the range of 3.5–4.2% [16]. This difference in DNA contents between X- and Y-bearing sperm can be exploited to effectively separate populations of X- and Y-sperm in mammals via high-speed flow cytometry [17]. The sample preparation requires sperm staining with the fluorochrome Hoechst 33342 [18], which is a vital stain that is able to penetrate the membrane of live sperm. After saturated binding of the dye to A–T rich regions of genomic DNA, fluorescence detection of small differences in relative DNA contents is possible [18]. Damaged sperm can be excluded by sorting through prior selective staining with FD#40, a red food dye [19]. The current high-speed sperm sorters operate at sample pressures up to 4.22 kg/cm2 and a droplet frequency of about 66 kHz, allowing identification of 30 000 events/s. Correctly oriented spermatozoa are eliminated from the system through the vibrating nozzle tip embedded in small fluid droplets. The UV-light excites the fluorescent dye, emitted light is collected by the 0 and 90 detectors and the electric signals are processed by a computer. Charged droplets disintegrate from the continuous stream and pass an electro-static field (3000 V), which allows separate collection of both the streams into tubes pre-filled with a medium. Sorted spermatozoa are subsequently washed from the sheath fluid by centrifugation and the remaining sperm pellet is extended in a suitable medium for further preservation [17, 20, 21]. The current models of modified high-speed flow sorters produce sexed sperm at the maximum rate of 20 million sorted spermatozoa per hour under optimal conditions http://www.cababstractsplus.org/cabreviews Wilfried A. Kues, Detlef Rath and Heiner Niemann with purities above 90%. Purity of sorted samples can be validated either by flow cytometrical reanalysis [22, 23], fluorescence in situ hybridization (FISH) [24, 25], or PCR [26]. The technology was first applied commercially in the UK in 2000, and approximately two million calves have been produced from insemination with sexed sperm since then (Rath and Johnson, in press). Although the sexing technology has become more efficient over the past decade, the number of sperm needed for regular inseminations (20 million sperm in cattle) cannot be flowsorted for large-scale commercial programmes. Thus, protocols for effective low-dose insemination have been developed. At least two million sperm are needed for commercial application of sex-separated bovine sperm [27]. However, this may be too low to obtain satisfactory pregnancy rates in light of the individual sperm differences of bulls to fertilize at such low concentrations [28]. This is even more pronounced in sorted-frozen spermatozoa that have a reduced life span, as indicated in thermotolerance tests [29]. In the pig, at least 50 million sperm are needed for deep intra-uterine AI to achieve acceptable pregnancy rates, thus preventing broader applications [30]. Reports on the fertility of sex-sorted semen are contradictory with that of some authors who find no decline of fertility and others reporting a reduced fertility after insemination with frozen sex-sorted spermatozoa [5, 31–35]. The lower fertility can be caused by physical and/or chemical stress during sorting, and/or can be caused by the low number of inseminated sperm. High laser intensity may cause more damage than lower laser intensity as shown for rabbit [36] and bull spermatozoa [37]. Employing the most advanced laser technology and replacing the water-cooled argon gas laser with a pulsed solid-state laser significantly reduces stress on sperm. The exposure of sperm to light pulses is much shorter than for the continuous argon laser beam. Preliminary observations with sorted ram spermatozoa using this new technology indicate increased fertility rates (WMC Maxwell, unpublished). Importantly, no changes were found in the frequency of endogenous DNA nicks after staining and UV light exposure, indicating that the dye is not genotoxic [38, 39]. The hydrodynamic pressure could also exert detrimental stress on the sorted sperm. Lowering the pressure from 2.59 to 2.07 mm Hg increased developmental rates of bovine in vitro fertilization (IVF) embryos produced from sex-sorted semen [40, 41]. Recently, we demonstrated for the first time that bovine IVF-blastocysts derived from sex-sorted spermatozoa showed an aberrant mRNA expression pattern of developmentally important genes such as glucose transporter 3 (Glut3) and glucose-6-phosphate dehydrogenase (G6PDX), compared with their counterparts derived from unsorted semen [42]. This indicates a longer lasting effect of sorting into preimplantation development. 3 Table 1 Birth of offspring (+) using sex sorted sperm with different fertilization techniques AI/DUI Cattle Pig Horse Sheep Goat Dolphins Rabbit Buffalo Elk Primates Cat + + + + + + + + + + ICSI IVF + + + + + + + + + AI/DUI, artificial insemination/deep intrauterine insemination; ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilization. Moreover, cleavage and blastocyst rates after IVF with sex-sorted spermatozoa were significantly lower than that in embryos produced with unsorted spermatozoa of the same ejaculate [43]. Earlier reports had indicated that the number of cell cycles is reduced [35] and timing of embryo development may be disturbed after insemination with sorted semen [33, 44, 45]. One approach to improve field results with sorted sperm would be to select sorted sperm populations that are specifically suited for successful insemination. With the aid of a 6-h thermotolerance test, sorted sperm populations were detected that showed improved fertility characteristics and could thus be better suited for insemination [29, 46]. The use of a novel sperm-preservation methodology (Sexcess1 ), based on a new extender supplemented with antioxidants, significantly increased lifespan and motility of bull spermatozoa. Pregnancy rates of heifers inseminated with sex-separated semen treated with Sexcess1 reached similar levels as for animals inseminated with non-sex-separated semen (73.6% sorted versus 76.7% controls; n=232) [47]. In the pig, it was shown that the media that had been supplemented with PSP (porcine seminal plasma) I/II heterodimers [48], which increases motility and mitochondrial activity [49], raised the proportion of viable sorted boar spermatozoa. More research is underway to improve bovine field results with sex-sorted semen by a better preselection and protection of spermatozoa during sorting and cryopreservation [50], pig [51–53], horse [54, 55]. IVF, intracytoplasmic sperm injection (ICSI) and intratubal insemination are promising approaches to adjust the sorting technology in pigs and horses, where the high number of viable spermatozoa needed for regular fertilization cannot be produced, even for deep intrauterine insemination of sorted spermatozoa (Table 1) [21, 48, 56–59]. In conclusion, flow-cytometry-based separation of sperm into X- and Y-chromosomes bearing populations http://www.cababstractsplus.org/cabreviews 4 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources Figure 1 Schematic drawing illustrating the use of somatic cloning for the production of transgenic animals. After the transfer of the transgenic donor cell into perivitelline space of the enucleated oocyte, both the components are fused usually by short, high-voltage pulses through the point of contact between the two cells. This is followed by activation of the reconstructed embryos, which is achieved either by short electrical pulses, or by brief exposure to chemical substances, such as ionomycin or dimethylaminopurin (DMAP), regulating the calcium influx into the complexes and/or the cell cycle. After a temporary in vitro culture period, the resulting blastocysts are transferred to synchronized recipient animals. already meets the requirements for field application of bovine semen when heifers are used and semen from preselected bulls is processed using the most advanced protocols. For yet-unknown reasons, cows have still lower conception rates after insemination with sex-sorted semen as compared with AI in heifers. Nevertheless, the current sexing technology is commercially available for cattle breeding and could evolve as a major tool towards the implementation of more efficient breeding and production schemes. More research is needed to allow commercial application of sex-sorted semen for other livestock species. Somatic Cloning of Farm Animals The technical aspects of nuclear transfer in mammals have already been established in the 1980s using blastomeres from early-stage embryos as donor cells [60, 61]. The assumption was that the closer the nuclear donor is developmentally to the early embryonic stage, the more successful nuclear transfer is likely to be. This was mainly based on earlier findings in frogs, in which cloning with embryonic cells was possible with the production of normal offspring, whereas cloning with adult cells was only compatible with the development into tadpoles [62]. This assumption dominated biology until the birth of ‘Dolly’ in 1996 – the first cloned sheep generated from an adult mammary gland cell [13]. Common somatic cloning protocols involve the following major technical steps (Figure 1): (1) Enucleation of the recipient oocyte: Oocytes at the metaphase II (MII) stage rather than any other developmental stage are the most appropriate recipients for the production of viable cloned mammalian embryos. These oocytes possess high levels of maturation promoting factor (MPF), which is thought to be critical for development of the reconstructed embryo [63]. In many domesticated species, oocytes can be obtained from abattoir ovaries, thus providing a potentially unlimited source of material for cloning experiments. Oocytes are enucleated by sucking or squeezing out a small portion of the oocyte cytoplasm, specifically the portion closely apposed to the first polar body, where the MII chromosomes are usually located. (2) Preparation and subzonal transfer of the donor cell: The entire intact donor cell, i.e. nucleus plus cytoplasm, is isolated from a cell culture dish by trypsin treatment and is inserted under the zona pellucida in intimate contact with the cytoplasmic membrane of the oocyte with the aid of an appropriate micropipette. Various somatic cells, including mammary epithelial cells, cumulus cells, oviductal cells, leucocytes, hepatocytes, granulosa cells, epithelial cells, myocytes, neurons, lymphocytes and germ cells, have successfully been used as donors for the production of cloned animals http://www.cababstractsplus.org/cabreviews Wilfried A. Kues, Detlef Rath and Heiner Niemann (3) (4) (5) (6) [63–66]. Fetal cells, specifically fibroblasts, have frequently been used in somatic cloning experiments, because they are thought to have less genetic damage and a higher proliferative capacity than adult somatic cells. One option to improve cloning efficiency could be using less-differentiated cells (foetal or stem cells) to minimize the complicated and error-prone reprogramming process. Somatic stem cells have been used successfully to give porcine blastocyst development and the birth of live piglets [67, 68]. In most experiments, donor cells are induced to exit the cell cycle by serum deprivation, which arrests cells at the G0/G1 cell cycle stage [69]. Serum deprivation of foetal fibroblasts for more than 72 h has been found to be associated with increased apoptosis rates [70, 71]. Specific cyclin-dependent kinases, such as roscovitin, have been reported to increase the efficiency of the cloning process, although final evidence in the form of a healthy offspring is lacking [63]. Nevertheless, unsynchronized somatic donor cells have been successfully used to clone offspring in mice and cattle [72, 73]. Fusion of the two components: Enucleated oocyte and donor cell are fused, usually by short, high-voltage pulses through a point of contact between the two cells. Activation of the reconstructed complex: Activation is achieved either by short electrical pulses, or by brief exposure to chemical substances such as ionomycin or dimethylaminopurin (DMAP), regulating the calcium influx into the complexes and/or the cell cycle. Temporary culture of the reconstructed embryos: Cloned embryos can be cultured in vitro to the blastocyst stage (5–7 days) to assess the initial developmental competence prior to transfer into a foster mother. Culture systems for ruminant embryos are quite advanced and allow the routine production of 30–40% blastocysts from in vitro-fertilized oocytes isolated from abattoir ovaries [74, 75]. Transfer to a foster mother or storage in liquid nitrogen: Bovine embryos at the morula and blastocyst stage can be transferred non-surgically to the uterine horns of synchronized recipients using established embryo-transfer procedures. In pigs, the activated nuclear transfer complexes are immediately transferred into the oviducts of the recipient because the in vitro culture systems for embryos are not yet as effective as in cattle. This requires a surgical intervention under general anaesthesia. To date, somatic nuclear transfer has been successful in 13 mammalian species (sheep [13], cow [72], mouse [76], goat [77], pig [78–80], rabbit [81], cat [82], horse [83], mule [84], rat [85], dog [86], ferret [87], wolf [88]) and also in the generation of cloned offspring from endangered species and breeds [89–92]. Attempts to preserve livestock genetic resources by somatic cell banking have 5 been reported [93, 94]. However, the typical success rate (live births) of mammalian somatic nuclear transfer is low: usually only 1–2%. Cattle seem to be an exception to this rule as levels of 15–20% can be reached [95]. Recently, we have obtained significant improvement of porcine cloning success by a better selection and an optimized treatment of the recipients, specifically by providing a 24 h asynchrony between the pre-ovulatory oviducts of the recipients and the reconstructed embryos. Presumably, this gave the embryos additional time to achieve the necessary level of nuclear reprogramming. This resulted in pregnancy rates of 80% and only slightly reduced litter size [96]. Pre- and post-natal development can be compromised and a variable proportion of the offspring in ruminants and mice show aberrant developmental patterns and increased perinatal mortality. These abnormalities include a wide range of symptoms, such as extended gestation length, oversized offspring, aberrant placenta, cardiovascular problems, respiratory defects, immunological deficiencies, problems with tendons, adult obesity, kidney and hepatic malfunctions, behavioural changes and a higher susceptibility to neonatal diseases and are summarized as ‘Large Offspring Syndrome’ (LOS) [97–101]. The incidence is stochastic and has not been correlated with aberrant expression of single genes or specific pathophysiology. A new term ‘Abnormal Offspring Syndrome’ (AOS) with sub classification according to the outcome of such pregnancies has recently been proposed to reflect better the broad spectrum of this pathological phenomenon [102]. The general assumption is that the underlying cause for these pathologies is insufficient or faulty reprogramming of the transferred somatic cell nucleus. However, a critical survey of the published literature on cloning of animals revealed that albeit the higher pre- and neonatal death rates of clones, most postnatal clones are healthy and develop normally [103, 104]. This is consistent with the finding that mammalian development is rather tolerant of minor epigenetic aberrations in the genome and subtle abnormalities in gene expression do not interfere with the survival of cloned animals [105]. It has become clear that once cloned offspring have survived the neonatal period and are approximately 6 months of age (cattle, sheep), they are not different from agematched controls with regard to numerous biochemical blood and urine parameters [106, 107], immune status [106], body score [106], somatotrophic axis [108], reproductive parameters [109], and yields and composition of milk [110]. No differences were found in meat and milk composition of bovine clones when compared with agematched counterparts; all parameters were within the normal range [111–113]. Similar findings were reported for cloned pigs [114]. There is a unanimous consent across various regulatory agencies around the world that food derived from cloned animals is safe and there is no scientific basis for questioning this [115], according http://www.cababstractsplus.org/cabreviews 6 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources Table 2 Important genomic features of selected livestock species and dog Species Genome size (109 bp) Chr. PC genes Pseudogenes RNA genes Cow Dog Chicken 3.25 2.39 1.05 29+X/Y 38+X/Y 32+W/Z 21 800 19 300 16 700 1296 1742 96 2496 2448 655 Chr., autosomal chromosome number and sex chromosomes; PC genes, number of identified protein coding genes; Pseudogenes, number of annotated pseudogenes; RNA genes, number of annotated RNA genes (RNA genes are not translated and act as structural or functional transcripts). Data retrieved from Ensembl. to the expert committee from the Japanese Ministry of Agriculture, Forestry and Fisheries (MAFF) [116], the FDA (Food and Drug Administration) [117] and EFSA (European Food Safety Agency) [118]. However, given the limited experience with somatic cloning (which has only been in general use since 1997) and the relatively long generation intervals in domestic animals, specific effects of cloning on longevity and senescence have not yet been fully assessed. Preliminary data indicate no pathology in serial cloning of mice and cattle [119, 120]. It has to be kept in mind that the clones frequently differ in a number of characteristics, because the two cytoplasms involved contain different maternal proteins, RNAs and mitochondrial DNAs. In addition, several epigenetic and environmental factors contribute to differences amongst groups of clones. Despite current limitations, somatic cloning has already emerged as an essential novel tool in basic biological research and holds great application perspectives, including the transgenic animal production. Genome Sequencing in Livestock Species Livestock genomics has followed the path that the human genome sequencing project has set, exploiting both strategy and technology. At the start of the Human Genome (HUGO) project, it was calculated that sequencing of one DNA base pair (bp) would cost around $1, thus sequence identification of the haploid human genome with approximately 3109 bp was calculated to cost US $3 billion. Over the past decade, impressive improvements in sequencing techniques and automatization have substantially reduced these costs. Today, sequencing of one mammalian genome would cost about US $100 000 and this price might be reduced even further through a better technology [121]. The sequencing and annotation of the human genome revealed a dramatically lower number of protein-coding genes than originally thought (20 000 versus 150 000 genes) [122], which corresponds well with estimates for other vertebrate species [123, 124]. The bovine genome is currently at the 4.0 read and 90% of the genes have been allocated to chromosomes [125] (Table 2). The chicken genomic map first became available in 2004 and is accessible from the International Chicken Genome Sequencing Consortium [126]. The dog genome followed in 2005 [127]. A draft sequence of the horse genome has been released recently (www.broad.mit.edu.mammals. horse). Open access to comprehensive genome information is provided by Ensembl (www.ensembl.org), University of California Santa Cruz (http://genome.ucsc.edu) or the National Center for Biotechnology Information (www.ncbi.org). The Ensembl project provides genome information for 47 species, including complete and pre-release low coverage of genomes of several livestock, other mammalian, vertebrate and non-vertebrate species. These genome data are valuable resources for the study of comparative genomics. Ensembl includes also polymorphism data, such as SNPs (single nucleotide polymorphisms) for 10 species. This database will be complemented by new biological data resources such as protein–DNA interaction (ChiP– chip) maps originating from the combination of genomewide chromatin immunoprecipitation (ChiP) with DNA microarray (chip) technology. The Ensembl browser allows visualization of genome data in an individualized manner. Figure 2 provides an example and shows a 100 kilobase (kb) region of bovine chromosome 2 including the locus of the myostatin (GDF8) gene. Loss of function mutations of this gene results in muscle overgrowth as observed in several beef cattle breeds [128– 130]. In Texel sheep, a gene mutation of the GDF8 gene produces a false microRNA target site, resulting in immediate transcript degradation and muscular hypertrophy [131]. In humans, a splice-site mutation of the GDF8 gene was found to cause pathological muscular hypertrophy [132]. This illustrates the great potential of comparative genomics using the Ensembl database. Direct access to genomic data and cell clones allows us to compare genomic regions of interest within populations or to design genetic modifications with a so far unprecedented speed and accuracy. For complex trait dissection, it is equally important to characterize the genetic variation within a population. Microsatellite markers and SNPs are the most commonly used polymorphisms for this purpose and have already revealed a surprisingly high degree of genetic variability in cattle [125]. SNP chips are rapidly being developed for farm animals. Affymetrix already offers a bovine 10 K SNP panel and Illuma has developed SNP chips for poultry, pigs and cattle with 7–25 K SNPs. http://www.cababstractsplus.org/cabreviews Wilfried A. Kues, Detlef Rath and Heiner Niemann 7 Figure 2 A 100 kb region of bovine chromosome 2 extracted from Ensembl. The region contains the gene locus of the myostatin gene (GDF8) encoding a major growth factor regulating muscle growth. Sequence information can be downloaded, and additional information such as GC ratio, CpG islands, SNP data and repeat elements can be assessed Genomic data can be used for improving selective breeding. Traditional methods of selection are based on selecting for quantitative traits using breeding value estimates calculated from measured traits, genetic covariances among traits and the pedigree relationships in populations. In contrast, selection using genomic information supplements phenotypic data with knowledge of the individual genotypes in a population for known relevant gene variants (=alleles). This process is significantly quicker and at the same time can easily be applied to traits with low heritability and slow genetic change. The prerequisite for genomic selection is knowledge of the trait data, the QTL (Quantitative Trait Locus) or the gene. Current SNP maps are not yet dense enough to allow full exploitation of the potential in effective breeding programmes. Another important use of genomic data is to study expression patterns (transcriptomics or proteomics) and the production of transgenic animals tailored for specific production niches or areas. Gene Expression Profiling of Preimplantation Embryos Complex organisms exploit a variety of regulatory mechanisms to extend the functionality of their genomes [133], including differential promoter activation, alternative RNA splicing, RNA modification, RNA editing, localization, translation and stability of RNA, expression of non-coding RNA, antisense RNA and microRNAs http://www.cababstractsplus.org/cabreviews 8 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources Figure 3 Transcriptome complexity and coverage of DNA expression arrays. Besides protein-coding transcripts, an unexpected large number of non-translated transcripts are expressed from the mammalian genome. Non-coding RNAs (ncRNA) include functional long ncRNAs such as XIST, AIR, CTN and PINK, and structural transcripts such as telomerase RNA (TERC), transfer RNA (tRNA) and ribosomal RNAs (rRNA). Small RNAs mainly act as regulatory units and include snoRNAs, microRNAs, siRNAs and piRNAs. The number of ncRNAs and small RNAs encoded within the genome is unknown. Recent transcriptomic studies suggest the presence of over 20 000 long ncRNAs and at least as many small regulatory RNAs [134–138]. These mechanisms mostly act at the RNA level and all together form the functional transcriptome of an organism (Figure 3). Tissue-specific transcriptomic profiles indicate important features of the regulatory process. During the past decade, efficient high-throughput methods have been developed for whole genome sequencing, transcriptome and proteome analyses [121, 139, 140]. The rapid improvements of DNA microarraytechnologies and the availability of multi-species sequence databases, such as the Gene Ontology database (www. geneontology.org) and the Kyoto Encyclopaedia of Genes and Genomes (KEGG) (www.genome.jp/kegg), have made it possible to compare gene expression profiles both within a single organism and across a wide variety of organisms on a large scale. These technologies are crucial to improve the efficiency and safety of oocyte-/embryorelated biotechniques, to facilitate the development of novel therapies based on regenerative medicine and to ensure the quality of biotechnologically derived products at the molecular level. In vitro culture of preimplantation embryos is frequently associated with significant alterations in chromatin configuration and gene expression [141–143]. In vitro production of bovine embryos is usually necessary to provide the large numbers of embryos needed for an array analysis in this species, but it is clear that the changes in embryonic transcription induced by the culture method itself seriously affect the transcriptional profile. The application of array technology to the analysis of preimplantation embryos poses specific challenges associated with the picogram levels of mRNA in a single embryo, the plasticity of the embryonic transcriptome and the difficulties in obtaining in vivo developing embryos. Gene expression patterns in mammalian embryos are more complex than those in most somatic cells because of the dramatic restructuring of paternal chromatin, the major onset of embryonic transcription, events related to the cellular differentiation at morula stage, blastocyst hatching and implantation. Embryo development is first dependent on maternally stored transcripts, which are gradually depleted until the embryo produces its own transcripts after embryonic genome activation [144]. The gene expression patterns and RNA stability in oocytes and early embryos prior to the activation of embryonic expression are dramatically different from what is seen after the major onset of embryonic transcription [145, 146]. The onset of embryonic gene transcription occurs at a species-specific time point; in mice, the major activation starts in late 1cell embryos; in humans and pigs, it occurs in 4-cell stages and in bovine embryos, is delayed until the 8–16-cell stage [147]. Given the late onset of this maternal-to-embryonic http://www.cababstractsplus.org/cabreviews Wilfried A. Kues, Detlef Rath and Heiner Niemann transition in bovine embryos, they provide an excellent model in which to study early reprogramming events in detail. Major epigenetic reorganization also occurs during preimplantation development. This includes DNA demethylation and methylation as well as targeted modifications of histone methylation and acetylation, all of which play a role in controlling chromatin remodelling and X-chromosome inactivation [148]. Transcriptome profiling by DNA array technology has been reported for human, mouse, bovine and porcine preimplantation embryos [145, 146, 147–158, reviewed in 159]. A study of the transcriptional profile of 2-cell mouse embryos included 4 000 genes from a cDNA-library and revealed many previously unknown transcripts, including mobile genetic elements that appear to be associated with activation of the embryonic genome [160]. The mRNA expression profile of porcine oocytes, 4- and 8-cell stages and blastocysts was studied with the aid of a 15 K cDNA array specific for porcine reproductive organs. The transcription profile of in vivo developing embryos was compared with that of in vitro produced 4-cell, 8-cell and blastocyst-stage embryos. This revealed 1409 and 1696 differentially expressed (in vivo/in vitro) genes at the 4- and 8-cell stages, respectively [155]. Studies of the bovine embryonic mRNA expression based on subtractive hybridization and cDNA arrays have identified differentially expressed genes between bovine oocytes derived from large or small follicles. A total of 21 upregulated genes were found out of 1000 clones [161]. Cross-species hybridization of a human cDNA array with bovine RNA, derived from rather large pools of >200 oocytes yielded a first expression profile for bovine oocytes before and after maturation. Approximately, 300 genes could positively be identified [152, 156, 162]. Recently, we have investigated the mRNA expression profiles of bovine oocytes and blastocysts by using a cross-species hybridization approach employing an array consisting of 15 529 human cDNAs (Ensembl chip) as probe, thus allowing the identification of conserved genes during human and bovine preimplantation developments [157]. The analysis revealed 419 genes that were expressed in both oocytes and blastocysts. The expression of 1342 genes was detected exclusively in the blastocyst, in contrast to 164 in the oocyte, including a significant number of novel genes. Typically, genes indicative for transcriptional and translational control, such as ELAVL4 and TACC3 were overexpressed in the oocyte, whereas genes indicative of numerous pathways were overexpressed in blastocysts. Transcripts implicated in chromatin remodelling were found in both oocytes (NASP SMARCA2, DNMT1) and blastocysts (H2AFY, HDAC7A). Expression patterns in bovine and human blastocysts were identical to a large extent [157]. A major advantage of this cross-species hybridization approach is that developmentally conserved novel and annotated marker genes could be identified for human and bovine oocytes and blastocysts. 9 Affymetrix commercially distributes a bovine genomic array which is based on the bovine genome sequence data and covers probe sets of approximately 23 000 transcripts and 19 000 UniGene clusters. The transcriptomes of bovine in vitro matured oocytes and in vitro-produced 8-cell stages were analysed by this Affymetrix bovine array and genes critically involved in major activation of the embryonic genome were determined [163, 164]. Approximately, 370 genes with functions in chromatin modification, apoptosis, signal transduction, metabolism and immune response were differentially expressed between oocytes and 8-cell stages, including DNMT1, DNMT2 and MeCP2, which were specifically up-regulated in 8-cell stages. Recently, we have hybridized RNA from in vivo-produced bovine oocytes and all stages of bovine preimplantation development till the blastocyst stage on the bovine Affymetrix array. For the first time, this analysis will yield a complete picture of mRNA expression in the processes of oocyte maturation, fertilization, embryonic activation and cell lineage differentiation in a large mammalian species (manuscript in preparation). Transgenic Animals in Agriculture Proof-of-principle that introduced recombinant DNA can result in phenotypic changes in mammals was shown in 1982 by the production of transgenic mice expressing a rat growth hormone [165]. The first transgenic livestock species were produced by microinjection of DNA into a pronucleus of fertilized eggs [14]. Despite numerous efforts, the production of transgenic farm animals is still hampered by low efficiencies and high cost. Transgenic farm animal production has been extensively reviewed by us in the past few years [12, 95, 166]. The first recombinant pharmaceutical protein (human antithrombin III, ATryn1 ) isolated from the milk of transgenic goats was approved in June 2006 by the European Medicine Agency (EMEA), clearly showing the great potential of this transgenic approach. Whereas the biomedical applications are quite advanced, agricultural application areas are lagging behind. Table 3 gives an overview on the various attempts to produce animals transgenic for agricultural traits. Prominent examples are: environmentally friendly pigs, in which phosphorus excretion is reduced by transgenic expression of the phytase gene [7], transgenic pigs with an altered pattern of polyunsaturated fatty acids in skeletal muscle, thus providing healthier pork [10, 169], cattle with resistance against Staphylococcus aureus mastitis by transgenic expression of lysostaphin restricted to the mammary gland [180], or cattle with a knockout of the prion gene thus rendering cattle non-susceptible to bovine spongiform encephalopathy (BSE) [11], alterations of the physicochemical properties of milk by overexpression of beta- and kappa-casein clearly underpinning the potential for improvements in the functional properties of bovine milk [178]. http://www.cababstractsplus.org/cabreviews 10 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources Table 3 Overview on successful transgenic livestock for agricultural production Transgenic trait Key molecule Construct Gene transfer method Species Reference Increased growth rate, less body fat Increased growth rate, less body fat Increased level of polyunsaturated fatty acids in pork Increased level of polyunsaturated fatty acids in pork Growth hormone (GH) hMT-pGH Microinjection Pig [167] Insulin-like growth factor-1 (IGF-1) Desaturase (from spinach) Desaturase (from Caenorhabditis elegans) Phytase a-lactalbumin mMT-hIGF-1 Microinjection Pig [168] maP2-FAD2 Microinjection Pig [169] CAGGS-hfat-1 Somatic cloning Pig [10] PSP-APPA Genomic bovine a-lactalbumin mMx1-Mx a,K-a,K Microinjection Microinjection Pig Pig [7] [170] Microinjection Microinjection [171] [172, 175] Ker-IGF-1 Microinjection Pig Pig, sheep Sheep Visna LTR-env Targeting vector (homologous recombination) b-lactoglobulin-SCD Genomic CSN2 CSN-CSN-3 a-s2cas-mLF Microinjection Somatic cloning Sheep Sheep [175] [176]1 Microinjection Somatic cloning Microinjection Goat Cattle [177] [178] Cattle [179] Ovine b-lactoglobulin lysostaphin Somatic cloning Cattle [180] Phosphate metabolism Milk composition (lactose increase) Influenza resistance Enhanced disease resistance Wool growth Visna virus resistance Ovine prion locus Milk fat composition Milk composition (increase of whey proteins) Milk composition (increase of lactoferrin) Staphylococcus aureus mastitis resistance Mx protein IgA Insulin-like growth factor-1 (IGF-1) Visna virus envelope Prion protein (PRNP) Stearoyl desaturase b-casein k-casein Human lactoferrin Lysostaphin [173, 174] 1 Animals dead shortly after birth (from [12]). With further refinements of the genetic maps many more agricultural applications will be developed. Further qualitative improvements may be derived from technologies that allow precise modifications of the genome, including targeted chromosomal integration by sitespecific DNA recombinases such as Cre or flippase (FLP), or methods that allow temporally and/or spatially controlled transgene expression [181]. However, it remains to be shown whether transgenic farm animals will ever be used to improve agricultural applications, considering the public anxiety against gene modification and the extraordinary high costs to establish transgenic lines. Also our current understanding of genome organization is fragmentary and needs to be augmented. Improved techniques for transgenesis are required for a more efficient production of transgenic animals and to achieve targeted expression. Successful experiments to improve transgenesis by lentiviral vectors [182, 183], to achieve conditional expression [184] and to exploit large genomic sequences [185] are important steps in this direction. A powerful tool is RNA interference, which allows the suppression of specific mRNAs [186, 187]. The potential of this endogenous regulatory mechanism is highlighted by a naturally occurring mutation in Texel sheep, which results in an illegitimate target site for endogenous microRNA in the myostatin (GDF8) transcript, resulting in rapid degradation of the mRNA and muscular hypertrophy [131]. Specific degradation of myostatin transcripts can be mimicked by short interfering RNAs (siRNAs), illustrating the potential of RNAi for breeding purposes. For a transient gene knockdown, synthetic siRNAs are transfected in cells or early embryos [188, 189]. For stable gene repression, the siRNA sequences must be incorporated into a gene construct. RNAi knockdown of porcine endogenous retroviruses (PERV) has been shown in porcine primary cells and cloned piglets [190, 191], as well as knockdown of the prion protein gene (PRNP) in cattle embryos [192]. Germline transmission of lentiviral siRNA has been shown in rats over three generations [193]. In contrast to knockout technologies, which require time-consuming breeding strategies to obtain homozygous knockout lines, RNAi can rapidly be integrated into existing breeding lines. The first transgenic animals to be commercially marketed are ornamental fishes expressing recombinant fluorescent proteins, which are sold for their fanciness. Transgenic medaka (Oryzias latipes, rice fish) with a fluorescent green colour [194] were approved for sale in Taiwan. Transgenic zebrafishes (Danio rerio) with several http://www.cababstractsplus.org/cabreviews Wilfried A. Kues, Detlef Rath and Heiner Niemann 11 fluorescent colours [195] are sold under the brand name Glofish1 in the USA [196]. The fluorescent fishes might act as ‘door-opener’ for gene modifications in other species. Recombinant DNA as New Vaccines Recombinant DNA techniques and the emerging genomic sequence information will be useful for novel applications, such as the development of a new class of vaccines. For DNA vaccination the transient presence of the coding sequence of a pathogen is sufficient to stimulate an immune response of the treated organism. The ongoing biodiversity genome-sequencing project (e.g. www. ncbi.org) facilitates access to sequence data of pathogenic organisms, and combined with current DNA synthesis methods DNA vaccines can be produced within a short period of time. The first DNA vaccine was approved by the US Department of Agriculture (USDA) in 2005 (www.aphis.usda.gov/lpa/news/2005/07/wnvdna_vs.html); the vaccine is designed to protect horses against West Nile Virus. The development of DNA vaccines is based on the finding that injection of plasmid DNA into skeletal muscle is compatible with the production of ectopic proteins [197]. The main advantages of DNA vaccines are: DNA plasmids can be easily and at low costs prepared in compliance with GMP standards, DNA plasmids are extremely stable in lyophilized form, design of the plasmids is flexible, unlike attenuated pathogens DNA plasmids bear no risk of pathogen formation, and DNAvaccinated animals can be discriminated from their infected counterparts [198, 199]. The principle of a DNA vaccine is that a gene sequence encoding for one or more protein epitopes of the pathogen is subcloned behind a strong eukaryotic promoter in a plasmid vector. The plasmid is amplified in a bacterial mass culture and then highly purified by ion exchange columns. The plasmid is then injected into muscle tissue, taken up by some muscle fibres and translocated into myonuclei. The eukaryotic promoter drives expression of pathogenic DNA sequences, resulting in the presentation of pathogen epitopes on the cell surface followed by triggering both a humoral and a cellular immune response of the vaccinated organism. Plasmid DNA was found to be degraded within a few weeks after intramuscular injection in pigs [200]. Since only partial sequences of the pathogen’s genome are used, no functional pathogen can be produced and vaccinated animals be discriminated from infected animals, which show antibody formation against all epitopes. Outlook Development in animal genomics have broadly followed the path that the human genome field has led, both in Figure 4 A perspective for molecular breeding based on genomic data and ES cells terms of completeness of data and in developing tools and applications as demonstrated by the recent availability of advanced drafts of the maps of the bovine, chicken, dog and horse genomes. The possibility of extending knowledge through deliberately engineering change in the genome is unique to animals and is currently being developed through the integration of advanced molecular tools and breeding technologies. However, the full realization of this exciting potential is handicapped by gaps in our understanding of embryo genomics and epigenetic mechanisms which are critical in the production of healthy offspring. The convergence of the recent advances in reproductive technologies with the tools of molecular biology opens a new dimension for animal breeding. Major prerequisites are the continuous refinement of reproductive biotechnologies and a rapid completion and refinement of livestock genomic maps. A critical role in this context is played by embryonic stem cells (ES cells). The availability of cells with true pluripotent properties in conjunction with exploitation of genomic data will allow molecular breeding programmes that are significantly faster and more effective than current programmes (Figure 4). Despite numerous efforts, no ES cells with germ line contribution have been established from mammals other than mouse. ES-like cells, i.e. cells that share several parameters with true ES cells, have been reported in several species [201]. The recent finding that the transgenic expression of four transcription factors (Oct4, Sox2, Klf4 and c-Myc) convert murine fibroblasts into pluripotent ES cells [202] will stimulate similar approaches in livestock (Figure 4). Genetically modified animals will play a growing role in the biomedical field. Agricultural application might be further down the track given the complexity of some of the economically important traits. The efficient use of domestic animals is urgently needed in light of the worldwide shortage of arable land and the ever increasing human population. Genomics offers great opportunities to improve livestock production in the developed world and advance the ‘livestock revolution’ in http://www.cababstractsplus.org/cabreviews 12 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources the developing world [203]. In addition to the already exploited biomedical potential, biotechnology in farm animals with a strong link to genomic technologies can make a significant contribution towards a more efficient, highly diversified and targeted, and thus sustainable animal production. The challenge for scientists and practitioners is to exploit the great potential of animal biotechnology for the benefits of the mankind. 14. Hammer RE, Pursel VG, Rexroad Jr CE, Wall RJ, Bolt DJ, Ebert KM, et al. Production of transgenic rabbits, sheep and pigs by microinjection. 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All rights reserved doi: 10.1111/j.1399-3089.2008.00442.x XENOTRANSPLANTATION Knockdown of porcine endogenous retrovirus (PERV) expression by PERV-specific shRNA in transgenic pigs Dieckhoff B, Petersen B, Kues WA., Kurth R, Niemann H, Denner J. Knockdown of porcine endogenous retrovirus (PERV) expression by PERV-specific shRNA in transgenic pigs. Xenotransplantation 2008; 15: 36–45. 2008 Blackwell Munksgaard Abstract: Background: Xenotransplantation using porcine cells, tissues or organs may be associated with the transmission of porcine endogenous retroviruses (PERVs). More than 50 viral copies have been identified in the pig genome and three different subtypes of PERV were released from pig cells, two of them were able to infect human cells in vitro. RNA interference is a promising option to inhibit PERV transmission. Methods: We recently selected an efficient si (small interfering) RNA corresponding to a highly conserved region in the PERV DNA, which is able to inhibit expression of all PERV subtypes in PERV-infected human cells as well as in primary pig cells. Pig fibroblasts were transfected using a lentiviral vector expressing a corresponding sh (short hairpin) RNA and transgenic pigs were produced by somatic nuclear transfer cloning. Integration of the vector was proven by PCR, expression of shRNA and PERV was studied by in-solution hybridization analysis and real-time RT PCR, respectively. Results: All seven born piglets had integrated the transgene. Expression of the shRNA was found in all tissues investigated and PERV expression was significantly inhibited when compared with wild-type control animals. Conclusion: This strategy may lead to animals compatible with PERV safe xenotransplantation. Introduction Xenotransplantation using porcine cells, tissues or organs may offer a potential solution for the shortage of allogeneic human organs [1]. Pigs are the most favored donor species for several reasons: (i) similar physiology and size of organs, (ii) unlimited availability, (iii), short generation interval, high number of progeny (up to 15 to 18 piglets); (iv) relatively low costs, (v) maintenance under high hygienic conditions is possible, i.e., specified pathogen-free (spf) breeding and gnotobiotic delivery is possible, (vi) tools for the genetic modification to reduce the immunogenicity are available and (vii) somatic cloning is possible and thus production of transgenic animals can be 36 Britta Dieckhoff,1 Bjçrn Petersen,2 Wilfried A. Kues,2 Reinhard Kurth,1 Heiner Niemann2 and Joachim Denner1 1 Robert Koch Institute, Berlin, 2Department of Biotechnology, Institute for Animal Science, Mariensee, Neustadt, Germany Key words: porcine endogenous retrovirus – RNA interference – shRNA – siRNA – transgenic pig – xenotransplantation Address reprint requests to Joachim Denner, PhD, Robert Koch Institute, Nordufer 20, 13353 Berlin, Germany (E-mail: [email protected]) Received 6 November 2007; Accepted 12 November 2007 significantly enhanced [2]. However, prior to the clinical use of porcine xenotransplants, three main hurdles have to be overcome: The immunological rejections, the physiological incompatibility and the risk of transmission of porcine pathogens. The premier immunological hurdle, the hyperacute rejection, which is due to the stimulation of the human complement system through the gal alpha 1,3 gal epitopes on the surface of porcine cells can be overcome in a clinically acceptable manner by either the production of Gal knock-out animals [3,4] or animals over-expressing human complement regulatory factors [5]. Specified pathogen-free breeding of pigs can prevent transmission of most porcine microbes. However, porcine endogenous retroviruses (PERVs) are Knockdown of PERV in pigs integrated in the porcine genome [6,7], and can be released as infectious particles from normal pig cells [8–10] and can infect human cells in vitro [11– 15]. The three replication-competent subtypes of PERVs mainly differ in their receptor-binding site of the envelope protein [6,16]. The subtypes PERVA and PERV-B are polytropic and able to infect human cells in vitro, whereas the ecotropic PERVC infects only porcine cells. Retroviruses are tumorigenic and may cause immunodeficiencies in infected hosts. The risk of PERV infection has to be carefully assessed specifically in view of the fact that the human immunodeficiency viruses HIV-1 and HIV-2, causing AIDS in humans, are the result of multiple trans-species transmissions of retroviruses from non-human primates [17,18]. However, in several clinical applications of porcine islet cells or ex vivo perfusions of pig liver cells such as bridging during acute liver failure, no virus transmission could be detected [19–22]. Likewise, in a cohort of 160 patients treated with various porcine tissues, no PERV transmission was observed [23,24]. Moreover, PERV transmission was not found after pig to non-human primate transplantation and PERV infection experiments [25–27]. However, the risk of PERV transmission associated with tumors or immunodeficiencies is still prevalent [28]. Removal of PERV sequences by knock-out technology would be the safest strategy, but may prove difficult due to the presence of numerous replication competent and defective proviruses capable of recombination and complementing each other. Different strategies for the prevention of PERV transmission have been developed including the selection of low-producer animals based on sensitive PERV assays [29–31], the generation of an antiviral vaccine [32], and the inhibition of PERV expression by RNA interference (RNAi) [33–35]. In these first experiments efficient short interfering RNAs (siRNAs) were selected and inhibition of virus expression was studied in PERV infected human cells [33] and primary pig cells [36]. The mechanism of RNAi is highly conserved among different biological systems, including plants, flies, worms, and mammals [37]. After processing by the ribonuclease III-like enzyme Dicer, double stranded RNA is cleaved into small double stranded fragments (21 to 25 nt) [38]. In the cytoplasm, these siRNAs interact with the RNA induced silencing complex, which recognizes the target mRNA, that is homologous to the sequence of the siRNA. Nucleases, which are part of the complex, degrade the target RNA and prevent protein translation [39,40]. Transfection of mammalian cells with longer dsRNA (>30 nt) induced an interferon response associated with an unspecific degradation of mRNA and finally apoptosis. The Dicer processing step can be bypassed by transfecting cells with synthetic siRNAs [41]. The amount of siRNAs within the cells is diluted out during cell division, thus limiting the time of gene silencing activity to approximately four to eight cell doublings [42,43]. Expression of sh (short hairpin) RNAs, that contain a short loop sequence linking the forward and reverse strand, can overcome this limitation. Defined shRNA without any cap- or polyAstructure can be expressed by polymerase-IIIdependent promoters such as the murine or human U6 snRNA- or the human RNase P (H1) RNA-promoters [44–47]. In addition, cells with stable chromosomal integration and permanent inhibition of target genes can be obtained if adequate selection markers are used. In addition, retroviral vectors may be used to integrate the siRNA producing vector [36]. The efficacy of RNAi has been investigated in vivo, until now mainly in mice, rats, and cattles [48–52]. Here, we show for the first time lentiviral vectormediated RNAi in pigs associated with a significant inhibition of PERV expression. Materials and methods Cell culture and lentiviral transfection Primary pig fibroblasts were transduced with the lentiviral vector pLVTHM-pol2 (Fig. 1). The vector expressed a shRNA corresponding to the viral pol2 sequence able to inhibit PERV expression efficiently [36]. Transduced cells were grown as described [53], selected by FACS using the expression of the reporter gene GFP and used in nuclear transfer. Generation of transgenic pig embryos by somatic nuclear transfer Transgenic pigs were generated by somatic nuclear transfer cloning using in vitro matured abattoir oocytes [54]. Briefly, oocytes were enucleated by removing the first polar body along with adjacent cytoplasm containing the metaphase plate. The donor cells were arrested at G0/G1 of the cell cycle by contact inhibition and serum starvation [DMEM + 0.5% fetal calf serum (FCS)] for 48 h. Prior to nuclear transfer, cells were treated for 10 min with trypsin/EDTA and centrifuged (200 g/3 min) twice in a 10 ml tube containing 5 ml phosphate-buffered saline and 100 ll FCS. The supernatant was removed and the cells were resuspended in Ca2+-free TL-HEPES medium. A small 37 Dieckhoff et al. Fig. 1. Localization of the primers specific for GFP and pol2 in the lentiviral vector pLVTHM-pol2 (LTR long terminal repeat, H1 polymerase III H1-RNA gene promotor, shRNA short hairpin RNA, SIN self-inactivating element, cPPT central polypurine tract, GFP green fluorescent protein, WPRE post-transcriptional element of woodchuck hepatitis virus, EF-1alfa elongation factor-1a promoter, tetO tetracycline operator, loxP locus of X-over of P1, recombination site of Cre recombinase for bacteriophage P1, the size of the amplicons is indicated). fibroblast from the positive cell clone was placed in the perivitelline space in close contact with the oocyte membrane to form a couplet. After cell transfer, fusion was induced in Ca2+-free SOR2 medium [0.25 m Sorbitol, 0.5 mm Mg-acetate, 0.1% bovine serum albumin (BSA)]. A single electrical pulse of 15 V for 100 ls (Eppendorf Multiporator, Eppendorf, Germany) between two electrodes was employed to fuse the membranes of the oocyte and the donor cell. The reconstructed embryos were activated in an electrical field of 1.0 kV/cm for 45 ls in SOR2 activation medium (0.25 m Sorbitol, 0.1 mm Ca-acetate, 0.5 mm Mg-acetate, 0.1% BSA) followed by incubation with 2 mm 6-dimethylaminopurine (DMAP; Sigma, Steinheim, Germany) in NCSU23 medium for 3 h prior to transfer to recipients. Synchronization of recipients and embryo transfer Peripuberal German Landrace gilts served as recipients. The gilts were synchronized by feeding 20 mg/pig Altrenogest (Regumate; Janssen-Cilag, Neuss, Germany) for 13 days followed by an injection of 1000 IU Pregnant Mare Serum Gonadotropine (PMSG; Intergonan, Intervet, Unterschleissheim, Germany) on the last day of Altrenogest feeding. Ovulations were induced by intramuscular injections of 500 IU human Chorion Gonadotropin (hCG; Ovogest, Intervet) 80 h after PMSG administration. Nineteen hours later, 115 reconstructed embryos were surgically transferred into the oviducts of one recipient via mid ventral laparatomy under general anesthesia induced and maintained with thiopental sodium (Trapanal, Altana, Konstanz, Germany). Maintenance of pregnancies was supported by administration of 1000 IU PMSG and 500 IU hCG on days 12 and 15 after surgery. Pregnancy was confirmed by ultrasound on days 25 and 35. 38 DNA isolation DNA was isolated from ear biopsies using a salt chloroform extraction method. Sixty micrograms tissue were incubated in 500 ll HOM buffer (160 mm saccharose, 80 mm EDTA, 100 mm Tris–HCl pH 8.0) (all reagents in this chapter from Roth, Karlsruhe, Germany) and 20 ll of a proteinase K solution (20 mg/ml; Invitrogen, Karlsruhe, Germany) at 60 C over night. After addition of 200 ll of a 4.5 m NaCl solution and 700 ll chloroform/isoamyl alcohol, DNA was extracted by centrifugation (10 min, 10 000 g, 4 C). The aqueous phase containing genomic DNA was mixed with 700 ll isopropanol and centrifuged as described above. After washing with 70% (v/v) ethanol the precipitated DNA was resuspended in 70 ll H2O and stored at )20 C until assayed. Detection of transgene Integration of the transgene was investigated by two independent PCR assays, using primers specific for (i) GFP (GFP for: 5¢-GATCACGAGACTAGCCTCGAGGT, GFP rev: 5¢-CCAGGATGTTGCCGTCCTC) and (ii) the shRNA expression cassette (pol2 for: 5¢-AACGCTGACGTCATCAAC, pol2 rev: 5¢-GGACGCTGACAAATTGAC) under the following temperature conditions: 95 C, 10 min; 35 cycles (95 C, 30 s; 45 C and 54 C respectively, 30 s; 72 C, 1 min); 72 C, 5 min. Isolation of siRNA and total RNA The siRNA and siRNA-depleted total RNA isolation from frozen tissues was carried out using the mirVana miRNA isolation Kit (Ambion, Huntingdon, UK) allowing the enrichment of RNAs Knockdown of PERV in pigs smaller than 200 nucleotides according to the manufacturer’s instructions and stored at )80 C until assayed. RNA probe construction For the in liquid hybridization a short 32P (GE Healthcare, Freiburg, Germany)-labeled RNA probe (29 nt) was generated by in vitro transcription using the mirVana miRNA Probe Construction Kit (Ambion). A DNA oligonucleotide with the reverse complement sequence of the target RNA was used as template for the in vitro transcription (pol2siRNA) and an additional T7 promotor sequence (5¢-CCTGTCTC-3¢) on it’s 3¢end was used (5¢-AGTCAATTTGTCAGCGTCCcctgtctc-3¢). The transcription reaction and the gel purification of the labeled probe were performed according to the manufacturer’s instructions. Visualization was carried out by phosphor-imaging (Imagine Plate Type Bas-III and FLA-2000; Fujifilm, Düsseldorf, Germany) with an exposition time of 30 to 60 s. Detection of siRNA Pol2-siRNA was detected with the mirVana miRNA Detection Kit (Ambion) according to the manufacture’s instructions, detection was carried out by phosphor-imaging with an exposure time of 3 to 7 min. One-step RT real-time PCR Quantitative real-time RT PCR was performed using the SuperScript One-Step RT-qPCR System with Platinum Taq DNA polymerase (Invitrogen) and the MX4000 thermocycler (Stratagene, La Jolla, CA, USA). A FAM-labeled probe (5¢FAM-AGAAGGGACCTTGGCAGACTTTCTBHQ1; Sigma) as well as primers specific for PERV gag, amplifying the viral full length RNA of all three subtypes (for: TCCAGGGCTCATAATTTGTC, rev: TGATGGCCATCCAACATCGA) were applied. PERV expression was normalized to the amount of total RNA as well as to the expression of the house-keeping genes glycerinaldehyd-3-phosphate-dehydrogenase (GAPDH), cyclophilin and hypoxanthine-guanine phosphoribosyltransferase (HPRT). A HEX-labeled probe and primers specific for porcine GAPDH (5¢-HEXCCACCAACCCCAGCAAGAGCACGC-BHQ1, for: 5¢-ACATGGCCTCCAAGGAGTAAGA, rev: GATCGAGTTGGGGCTGTGACT) (Operon, Cologne, Germany), a Cy5-labeled probe and primers specific for porcine cyclophilin (5¢-Cy5TGCCAGGGTGGTGACTTCACACGCC-BHQ2, for: 5¢-TGCTTTCACAGAATAATTCCAGGATTTA, rev: 5¢-GACTTGCCACCAGTGCCATTA, Operon), as well as a FAM-labeled probe and primers specific for porcine HPRT (FAM-ATCGCCCGTTGACTGGTCATTACAGTAGCT-BHQ1, for: 5¢-GTGATAGATCCATTCCTATGACTGTAGA, rev: 5¢-TGAGAGA TCATCTCCACCAATTACTT were used [55]. For each reaction, 50 ng total RNA and the following temperature conditions were used: 50 C, 15 min; 95 C, 2 min; 45 cycles (95 C, 15 s; 54 C, 30 s). PERV expression in each organ was normalized to porcine GAPDH, porcine cyclophilin and total RNA, respectively, and compared with the expression in the corresponding organ of the control animal. Samples from control and experimental animals were always run together. Data were analyzed using the DDCTmethod [56]. Western blot analyzes Western blot analyzes using sera specific for p15E and p27Gag of PERV were performed as described [36,57]. Results shRNA-mediated inhibition of PERV expression in primary fibroblasts Porcine fetal fibroblasts (primary cell line: P1 F10) were transduced using the lentiviral vector pLVTHM-pol2 (Fig. 1) expressing the PERV-specific shRNA pol2. In order to analyze the shRNAmediated inhibition of PERV-mRNA expression, total RNA was isolated from transduced and nontransduced fibroblasts and one-step RT real-time PCR was performed. PERV full-length mRNA expression was inhibited by 94.8% in fetal fibroblasts P1 F10 transduced with pLVTHM-pol2 as described previously [36]. The inhibition of PERV expression in transduced cells was stable and remained at 95% over months. Somatic cell nuclear transfer and the production of shRNA transgenic pigs and control animals The pLVTHM-pol2-transduced fibroblasts were cultured in vitro prior to being used for the production of transgenic pigs via somatic cell nuclear transfer. After activation, 115 cloned embryos were transferred surgically into the oviducts of one synchronized recipient. Pregnancy was confirmed by ultrasound scanning on days 25 and 35 after embryo transfer. After 116 days of 39 Dieckhoff et al. gestation seven piglets were born; one of them was stillborn. None of the piglets showed any malformations. Mean birth weight was 1.3 kg which is similar to that of non-transgenic piglets in our pig facility. Ear biopsies were obtained from six piglets and frozen in liquid nitrogen. Four piglets died soon after birth due to agalactia of the sow. Following repeated application of 2 ml oxytocin (Oxytocin 10 IE/ml; Pharma Partner, Hamburg, Germany) lactation was reinitiated and two piglets survived. They were killed by barbiturate overdose at day 3, and samples of heart, lungs, spleen, liver, kidney, pancreas as well as muscle were taken and frozen in liquid nitrogen for further analysis. Control animals were produced by nuclear transfer using primary fibroblasts from non-transduced porcine fibroblasts P1 F10. At day 89 of gestation, organs of five animals were obtained by Caesarian section, the fetuses had a mean weight of 1.1 kg. Fig. 3. Expression of the PERV-specific pol2-shRNA in different tissues of piglets no. 6 and 7. As positive control pLVTHM-pol2-transduced PK-15 cells and as negative controls pLVTHM-transduced and non-transduced PK-15 cells were used. Internal negative controls: no target RNA (T)/ RNase (the single stranded probe was degraded by RNase treatment), no target RNA/ no RNase (the full-length probe of 29 nt was not digested). Expression of the pol2-shRNA in vivo Detection of vector integration To detect the presence of the transgene, the lentiviral vector pLVTHM-pol2, in the genome of the piglets, PCRs were performed with primers specific for gfp and specific for the shRNA expression cassette (Fig. 1). All of the six tested piglets were positive for gfp (piglet 1 was not tested) and the shRNA expression cassette (all animals were tested positive), respectively (Fig. 2). For analysis of the expression of the pol2-shRNA in different organs, small RNA molecules were isolated from the organs of piglets no. 6 and 7 and used for Northern solution hybridization with a 32 P-labeled short RNA probe corresponding the shRNA sequence. Expression of pol2-shRNA was detected in all organs, including heart, lung, spleen, liver, kidney, muscle, and pancreas (Fig. 3). Inhibition of PERV expression in different tissues Fig. 2. (A) Integration of the lentiviral vector pLVTHM-pol2 in six piglets demonstrated by PCR analysis using primers specific for GFP, and (B) primers specific for the shRNA expression cassette, positive control (+): vector pLVTHMpol2, negative control ()): DNA of non-transduced porcine fibroblasts. N.t. not tested. 40 Total RNA was isolated from different organs of piglets no. 6 and 7 and analyzed using one-step RT real-time PCR. As control, total RNA isolated from the corresponding organs taken from five control, non-transgenic pigs was used. Expression of PERV in different organs of the shRNAtransgenic and the non-transgenic control animals was highest in the spleen and lower expression was found in the liver. Due to the differences in PERV expression related to individual organs, PERV expression was compared in shRNA-transgenic and non-transgenic animals on a per organ basis. The expression levels of PERV in each organ of the control pigs were averaged and set 100%. PERV-expression in the corresponding organs from the shRNA-pol2 transgenic piglets was related to the expression in the control organ. Data were normalized to the amount of total RNA or the expression of the house-keeping genes GAPDH, cyclophilin, and HPRT. In all organs of the pol2-shRNA transgenic piglets no. 6 and 7 PERV expression was significantly inhibited by up to 94% (Fig. 4). Irrespective of the type of Knockdown of PERV in pigs Fig. 4. Inhibition of PERV expression in different tissues of the pol2-shRNA transgenic piglets (p) no. 6 and 7. PERV expression in each organ of control non-transgenic animals (c) measured by one-step RT real-time PCR was set 100% and compared with PERV expression in the corresponding organ of the transgenic animals. In case of control animals the standard deviation is based on the measurement of PERV expression in organs from five different animals, measured in triplicate; in case of transgenic animals the standard deviation is based on the measurement of one organ measured in triplicate in two assays. Expression was normalized to the amount of total RNA, measured by one-step RT real-time PCR. normalization (total RNA amount or housekeeping genes GAPDH, cyclophilin, and HPRT) similar expression patterns were observed. Noteworthy, expression of PERV in organs of the control animals was similar to the expression of PERV in adult animals. Preliminary data showed an expression of 2446 copies of PERV/ng in the hearts from five control animals, 500 and 492 in the hearts of the transgenic piglets 6 and 7, respectively. 4564 copies/ng were found in the control lungs vs. 1337 and 936 in the lungs of the transgenic piglets 6 and 7. In the control liver 1614 copies/ng were detected vs. 166 and 328 in the livers of the transgenic piglets, in the spleens 34068 vs. 1908 and 2419, in the kidneys 2532 vs. 975 and 390, showing the efficacy of the RNAi in vivo. Undedectibility of PERV protein expression and virus release When PERV protein expression was studied using Western blot analyses of homogenates from different tissues using antisera specific for different PERV proteins (p15E, p27Gag), no PERV protein expression was observed, neither in the non-transgenic controls nor in the transgenic animals (not shown). Discussion Results of the present study demonstrate a strong and long lasting inhibition of PERV mRNA expression in primary porcine fibroblasts using a lentiviral vector system expressing a PERV-specific shRNA and use in somatic cell nuclear transfer. This is the first report demonstrating shRNAmediated reduction of PERV expression in shRNA-transgenic pigs in vivo. The strategy applied here could lead to microbiological safer porcine xenotransplants and is therefore of considerable medical importance. More than fifty copies of porcine endogenous retroviruses are integrated in the porcine genome [6,58,59], rendering conventional gene-knockout approaches not feasible and making RNAi a promising alternative. The RNAi approach has already been used for the in vitro inhibition of HIV [60–62] and other viral pathogens like hepatitis B virus (HBV) [63,64], Coxsackie virus B3 [65], West Nile virus [66], and transmissible gastroenteritis virus [51]. Furthermore, RNAi-mediated reduced HBV expression has been demonstrated in mice in vivo [67,68] by murine hydronamic tail vene injection. Long-term inhibition of HBV was achieved by adeno-associated vector-mediated RNAi [69]. RNAi was also induced by lentiviral gene transfer in transgenic mice with a long-term inhibition of the target gene [70]. In contrast, HIV1 escapes from RNAi-mediated inhibition not only through nucleotide substitutions or deletions in the siRNA target sequence, but also through mutations that alter the secondary RNA structure [71]. Resistance and/or escape from the inhibitory effects of RNAi are impossible in the case of PERV, since its genome behaves like a cellular gene. 41 Dieckhoff et al. In future experiments, the number of integrated shRNA vectors as well as the influence on the expression of PERV during life time and the persistence of the shRNA expression in the next generation will be analyzed. The physiological role of endogenous retroviruses is unknown. Our results indicate that the siRNA expression did not compromise the fetal development because the siRNA transgenic piglets were born after a normal gestation period, with a normal birth weight and without malformations. Due to the sequence specificity of the RNAi mechanism and the fact that PERV is a retroviral sequence, off-target effects resulting in gene silencing of vital cellular proteins are highly unlikely. Some RNAi off-target effects have recently been reported when interfering with the expression of cellular target genes [72–75]. Due to the early death of the pol2-shRNA-transgenic piglets caused by agalactia of the sow, long-term observations of the inhibitory effect could not yet be made. In transgenic mice expression of the Neil1 gene, a DNA N-glycosylase, was inhibited by shRNA over an extended period of time in all organs [76]. The detection of the inhibitory effect of the pol2shRNA on PERV expression in vivo requires a suitable control, especially in the presence of a very low expression of the PERV in the non-transgenic founder pigs. In addition, PERV expression varies in different pig strains [9,31] as well as in different tissues and organs of one individuum [77,78]. Thus detailed analysis of non-transgenic pigs of the same strain provides only limited insight into the RNAi effects. To provide an appropriate control, pigs were produced by somatic cell nuclear transfer using the non-transfected counterparts of the identical primary cell isolate. This allowed us to compare PERV expression between animals and organs isolated from different donors and this comparison revealed a significant reduction of PERV expression in the siRNA transgenic pigs. Expression data were normalized both to the amount of total RNA and to the expression of different house-keeping genes such as GAPDH, cyclophilin, and HPRT. HPRT is considered to be a suitable reference gene for the analyses of gene expression in different porcine tissues and immune cells [79,80]. Numerous studies on GAPDH expression in humans and rodents showed changes related to different experimental conditions. In addition, the existence of pseudogenes for HPRT [81] and GAPDH [82] complicates the use of these genes as standards in RT-PCR reactions. However, although the expression of all house-keeping genes used here was different in different organs, normalization of PERV expression according to the 42 expression of each of these house-keeping genes showed a similar inhibitory effect of PERV expression up to 95%. Based on the low expression of viral RNA also a low expression of PERV proteins was expected. In Western blot assays using sera specific for p15E and p27Gag of PERV, no protein expression was observed, neither in the non-transgenic controls nor in the transgenic animals. Using the same assays PERV protein expression was also not detected in primary PBMCs from different pig strains such as German landrace, Duroc, Schwäbisch Hällisch, German large white as well as crossbreeds. In PK-15 cells, producing detectable amounts of infectious virus particles, expression of p27Gag was detectable, but still very low and this expression was reduced by RNAi nearly to 100% [36]. Higher expression of p27Gag protein was observed in human 293 cells infected with a high titer strain of PERV, which was only partially inhibited by RNAi [33]. A higher expression of PERV proteins was also observed in pig melanomas and in melanoma cell lines [57]. The absence of viral proteins in non-transgenic and transgenic pigs suggested the absence of PERV release. However, the low sensitivity of the Western blot assay (detection limit: 50 ng for p15E and 100 ng for p27Gag [57] left questions open. Improving the assays using monoclonal antibodies, developing a sensitive immunohistochemistry analysis or using a sensitive assay to detect RT activity in the plasma, such as the PERT (productenhanced reverse transcriptase) assay [83] may help to compare protein expression and virus release in transgenic and non-transgenic animals more correctly. To increase the safety of porcine xenotransplants, multiple shRNAs corresponding to different regions of the PERV genome shall be used. The advantage of the RNAi strategy is, that the expression of all PERVs will be inhibited and the likelihood of recombinations or complementation is further decreased. Selection of PERV lowproducer pigs using methods described earlier [9,31] together with the production of shRNAtransgenic animals may reduce PERV expression in cells and organs and thus increase the viral safety of porcine xenografts. Acknowledgments The authors gratefully acknowledge funding by DFG within the TransRegio research group 535. 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