Embryonic stem cells as genetic tool

Embryonic stem cells as genetic tool
Ralf Kühn, GSF Research Center, Institute for Developmental Genetics, Neuherberg
Embryonic stem (ES) cells are pluripotent, primary cell lines derived from the inner cell mass
(ICM) of mouse blastocysts. Under appropriate conditions, the embryonic developmental potential
of ES cells can be maintained following genetic manipulations and after many passages of in vitro
culture. Upon reintroduction into preimplantation embryos ES cells contribute to all embryonic
tissues in chimaeric mice, including the gametes. Genetic modifications introduced into ES cells
through homologous recombination or random insertional mutagenesis can thereby transmitted
through the germline to establish mutant mouse strains. ES cells as tool for reverse mouse genetics
enabled the construction of ~3.000 knockout mouse lines and gene trap libraries of mutant ES cell
clones that cover one third of all mouse genes. Furthermore, ES cells can be differentiated in vitro
into various cell types, including male or female germ cells, and allow to study signal requirements
for lineage determination as well as the repair of damaged tissue in vivo.
Presently we develop an ES cell based approach to insert conditional RNAi expression vectors into
a defined genomic location through site-specific recombination. This approach may allow to
produce knockdown mutants at larger scale and within less time as presently achieved with gene
knockout methodology. Through the use of F1 hybrid ES cells and tetraploid embryo
complementation completely ES cell-derived mice can be obtained at an efficiency of 5%. These
mice can be directly used for an initial phenotype assessment in comparison to wildtype F1
controls. For follow up studies larger numbers of male and female knockdown mice can raised by
natural mating.
© Lehrstuhl für Physiologie,
Letzte Änderung:28.09.2005, Renate Schöpf