How to construct transgenic mice
Sandra Beer-Hammer
Autumn School 2010
Bad Schandau
Methods
• additional genetic information
transgenic mouse line
• gene inactivation
Generation of Transgenic Mice
holding
pipette
cloned
gene
holding
pipette
cloned
gene
1-cell embryos
at pronuclear stage
injection
pipette
1-cell embryos
at pronuclear stage
injection
pipette
Microinjection of DNA
Holding
Injection
pipette
Holding
pipette
Embryo
(1-cell stadium)
Construction of DNA and Expression Control
T- and B-cell
T- and B-cell
specific promotor
Gene Deficient Mice „knock-out“
-/-Gene Targeting Method - Overview
Targeting Construct - Design
LT R: Genomic locus β Targeting vector E probe A E 17 kb E neo HSV-TKA
A X exon 1 2 3 4 5 6 7 8 9 10 vector Targeted allele E1 kB
E E neo probe A 4 kbNeo:
positive selection
Targeting Verification
E14
kB
17.0
w
t
+
/-4.0
Southern Blot
Isolation of Blastocysts
Injection of ES Cells
Holding
pipette
Blastocyst
(2.5 days)
Injection pipette
with ES cells
Testing of Germline Transmission
E14
mice
kB
w
t
w
t
+
/-+
/-Aguti mice carry
one allele of the
mutated gene
kB
17.0
4.0
Verification of KO
+
/+
+
/--/
-2.0
kB
LTβ
β
β
βR
2.0
1.2
LTβ
β
β
βR
GAPDH
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.)
Organ Size and Morphology
Immune Cell Subpopulations
Immune Response in vivo
TNP-Ficoll: IgG2a
d0
d4
d7 d14 d21
O
D
4
0
5
n
m
0,03 0,06 0,09 0,12 0,15*
**
TNP-Ficoll: IgG3
d0
d4
d7 d14 d21
O
D
4
0
5
n
m
0,03 0,06 0,09 0,12 0,15*
TNP-Ficoll: IgM
d0
d4
d7 d14 d21
O
D
4
0
5
n
m
0.1 0.2 0.3 0.4 0.5 0.6*
***
*
***
TNP-CGG: IgG1
TNP-CGG: IgG2a
TNP-CGG: IgM
d0 d4 d7 d14 d21
O
D
4
0
5
n
m
0.1 0.2 0.3 0.4 0.5 0.6 0.7*
d0 d4 d7 d14 d21
O
D
4
0
5
n
m
0.1 0.2 0.3 0.4 0.5 0.6*
*
**
TNP-CGG: IgG1
TNP-CGG: IgG2a
TNP-CGG: IgM
d0
d4
d7 d14 d21
O
D
4
0
5
n
m
0.1 0.2 0.3 0.4 KO WTImmune Tolerance
T
ra
n
sp
la
n
t
su
rv
iv
a
l
(%
)
80
100
H-2
bH-2
b, wt (n=5)
H-2
bxdH-2
b, wt (n=8)
bxd bH-2
bxdH-2
bdays
10
20
30
40
50
60
T
ra
n
sp
la
n
t
su
rv
iv
a
l
(%
)
20
40
60
H-2
H-2 , wt (n=8)
H-2
bxdH-2
b, sly-/- (n=10)
Conditional Gene-Targeting
neo neo
Procaryotic Rekombinase Systems
neo
neo
transient Cre (Frt, Flrt) Expression
conditional gene targeting
complete / multiple deletions
(long distance deletions)
Cre-mediated Deletion in vivo
Cre
X
Cre mouse (deleter or
Gene-targeted mouse
Cre
