How to construct transgenic mice

How to construct transgenic mice
Sandra Beer-Hammer
Autumn School 2010
Bad Schandau
Pharmakologie und Experimentelle Therapie (APET)
Methods
• additional genetic information
transgenic mouse line
• gene inactivation
gene-deficient „knockout“ mouse line
Jak2 -/Seite 2
Pharmakologie und Experimentelle Therapie (APET)
Transgenic Mice
Seite 3
Pharmakologie und Experimentelle Therapie (APET)
Generation of Transgenic Mice
holding
pipette
pipette
1-cell embryos
at
at pronuclear
pronuclear stage
stage
Seite 4
cloned
gene
gene
injection
pipette
pipette
Pharmakologie und Experimentelle Therapie (APET)
Microinjection of DNA
Injection
pipette
Holding
pipette
Embryo
(1-cell stadium)
Seite 5
Pharmakologie und Experimentelle Therapie (APET)
Construction of DNA and Expression Control
T- and B-cell
specific promotor
(B. Iritani, 1997)
Seite 6
Pharmakologie und Experimentelle Therapie (APET)
Protein Expression in T and B Cells
Seite 7
Pharmakologie und Experimentelle Therapie (APET)
Gene Deficient Mice „knock-out“
Jak2 -/-
Seite 8
Pharmakologie und Experimentelle Therapie (APET)
Gene Targeting Method - Overview
from Capecchi, 1998
Seite 9
Pharmakologie und Experimentelle Therapie (APET)
Targeting Construct - Design
A
17 kb
E
LTβR:
Genomic locus
probe A
A
exon
1 2
X
3 4
5 6
E
7 8
9
10
E
neo
HSV-TK
Targeting
vector
4 kb
E
E
probe A
E
neo
Targeted
allele
1 kB
Neo:
HSV-TK:
Seite 10
positive selection
negative selection
Pharmakologie und Experimentelle Therapie (APET)
Transfection of ES Cells
Seite 11
Pharmakologie und Experimentelle Therapie (APET)
Targeting Verification
+/-
wt
E14
kB
17.0
4.0
Southern Blot
Seite 12
Pharmakologie und Experimentelle Therapie (APET)
Isolation of Blastocysts
(C57/BL6)
Seite 13
Pharmakologie und Experimentelle Therapie (APET)
Injection of ES Cells
Holding
pipette
Blastocyst
(2.5 days)
Seite 14
Injection pipette
with ES cells
Pharmakologie und Experimentelle Therapie (APET)
Chimeric Offspring
Seite 15
Pharmakologie und Experimentelle Therapie (APET)
Testing of Germline Transmission
Aguti mice carry
one allele of the
mutated gene
+/-
mice
wt
+/-
wt
E14
kB
17.0
4.0
Chimeric mice are mated with
C57/BL6 mice
Seite 16
Southern Blot
Pharmakologie und Experimentelle Therapie (APET)
-/-
+/-
+/+
Verification of KO
kB
2.0
LTβ
βR
1.2
GAPDH
Northern Blot
Seite 17
Western Blot
Pharmakologie und Experimentelle Therapie (APET)
Phenotype Analyses
- Organ size and morphology
- Immune cell subpopulations (frequency, function)
- Immune response in vitro (Proliferation, Cytokine production)
- Immune response in vivo (Immunisation with Ficoll, CGG)
- Defense of infections (Listeria, Toxoplasma, etc.)
- Immune tolerance (Transplantation of Skin, Heart, Kidney)
- Autoimmunity and Allergy (DTH, Asthma, etc.)
- etc.
Seite 18
Pharmakologie und Experimentelle Therapie (APET)
Organ Size and Morphology
Impaired Organogenesis of LNs without LTβR
Seite 19
Pharmakologie und Experimentelle Therapie (APET)
Immune Cell Subpopulations
Reduced DP and SP Thymocytes in SLy1-deficient Mice
Seite 20
Pharmakologie und Experimentelle Therapie (APET)
Immune Response in vivo
0,12
0,09
0,06
0,03
*
**
d0 d4 d7 d14 d21
d0 d4 d7 d14 d21
TNP-CGG: IgM
TNP-CGG: IgG1
0.4
OD 405nm
*
0,15
KO
WT
0.3
0.2
0.1
d0
d4 d7 d14 d21
0.7
0.6
0.5
0.4
0.3
0.2
0.1
**
*
0,15
0,12
*
0,09
0,06
0,03
d0 d4 d7 d14 d21
TNP-CGG: IgG2a
0.6
OD 405nm
***
*
OD 405nm
***
OD 405nm
OD 405nm
0.6
0.5
0.4
0.3
0.2
0.1
TNP-Ficoll: IgG3
OD 405nm
TNP-Ficoll: IgG2a
TNP-Ficoll: IgM
0.5
*
0.4
*
0.3
0.2
0.1
d0 d4 d7 d14 d21
d0 d4 d7 d14 d21
Impaired Antibody Production in SLy1-deficient Mice
Seite 21
Pharmakologie und Experimentelle Therapie (APET)
Immune Tolerance
Transplant survival (%)
H-2bxd
H-2b
100
H-2b H-2b, wt (n=5)
H-2bxd H-2b, wt (n=8)
H-2bxd H-2b, sly-/- (n=10)
80
60
40
20
10
20
30
40
50
60
days
Increased Allograft Survival in SLy1-deficient Mice
Seite 22
Pharmakologie und Experimentelle Therapie (APET)
Conditional Gene-Targeting
neo
genetically modified ES cell
Seite 23
Pharmakologie und Experimentelle Therapie (APET)
Procaryotic Rekombinase Systems
neo
transient Cre (Frt, Flrt) Expression
complete / multiple deletions
conditional gene targeting
(long distance deletions)
Seite 24
Pharmakologie und Experimentelle Therapie (APET)
Cre-mediated Deletion in vivo
X
Cre
Gene-targeted mouse
Cre mouse (deleter or
tissue/cell-specific)
Cre
constitutive or inducible
deletion of gene of interest
Seite 25
Pharmakologie und Experimentelle Therapie (APET)
The Cre-Zoo (constitutive or inducible)
Lck
DN T cells
CD4
DP T cells
FoxP3
Treg cell
CD19
B cells
Mb1
early B cells
CD45
Lymphocytes
F4/80
Macrophages, Kupffer cells
CD11c
DCs
Albumin
Hepatocytes
Tie1/2
Endothelial cells
etc.
Seite 26
Pharmakologie und Experimentelle Therapie (APET)
BAC Transgenic (or knock-out) Mice
Seite 27
Pharmakologie und Experimentelle Therapie (APET)
BAC Transgenic (or knock-out) Mice
Fluorescent proteins
Light-inducible
cation channel
Cre expression
Cre inducible DTR
Inducible Cre
Inducible gene expression
LacZ expression
Light-inducible
anion channel
From Johansson, Genesis 2010
Seite 28
Pharmakologie und Experimentelle Therapie (APET)
Find the Right BAC (bacterial artificial chromosome
www.ensembl.org
From Johansson, Genesis 2010
Seite 29
Pharmakologie und Experimentelle Therapie (APET)
BAC Transgenic Mice
Purify DNA and
inject into pronucleus
From Johansson, Genesis 2010
Seite 30
Pharmakologie und Experimentelle Therapie (APET)
ET cloning versus Recombineering
From Johansson, Genesis 2010
Seite 31
Pharmakologie und Experimentelle Therapie (APET)
ET cloning versus Recombineering
From Johansson, Genesis 2010
Seite 32
Pharmakologie und Experimentelle Therapie (APET)
BAC knock-out
Seite 33
Pharmakologie und Experimentelle Therapie (APET)
BAC knock-out – Method Overview I
Creating the targeting vector with homologues recombination in E. coli
1.)
pBAD
WI1
electroporate
28
28°°C
2.)
NEOr
3.)
electroporate
pBAD
WI1
Picking Kana/Chloramph resistant clones / check by PCR + DNA restriction
Seite 34
Pharmakologie und Experimentelle Therapie (APET)
BAC Knock-out – Method Overview II
4.)
pBAD
WI1
electroporate
+NEO
28
28°°C
5.)
Thymidine kinase
electroporate
pBAD
WI1
+NEO
6.)
Seite 35
Picking Amp/Kana/Chloramph resistant clones / check by PCR + DNA
restriction
Pharmakologie und Experimentelle Therapie (APET)
Verification of Modified BAC
Purify DNA and
electroporate into ES cells
Seite 36
Pharmakologie und Experimentelle Therapie (APET)