Genetic manipulation of mouse

1
Genetic manipulation of mouse
Knockout
Mouse embryonic stem (ES) cells
Fertilized egg
Inner cell mass
trophoblast
Mouse embryo (13.5 days)
Blastocyst (3.5 days)
Media (leukemia inhibitory factor, LIF)
ES colonies
Mouse embryonic fibroblast (MEF)
2
Generation of knockout (targeting) construct
Gene to disrupt
genome
gene
3 homology
5 homology
neo
tk
Thymidine kinase gene
Neomycin resistance gene
Knockout construct
3
Homologous recombination
Knockout construct
transfection
gene
genome
neo
construct
tk
neo
genome
gene
construct
tk
(lost)
4
Random integration
neo
construct
tk
genome
Random DNA break
Nonhomologous site
neo
tk
5
Drug selection
G418s
Random integration
neo
tk
No integration
gancyclovirs
neo
Homologous integration
G418r, Gancyclovirr
6
Screen for homologous integration
(R) restriction enzyme site
ES colonies
R
R
gene
germline
R
R
R
neo
knockout
Pick and grow up Individual colony
Different restriction digest pattern
Analyze DNA
7
Inject knockout ES cell into blastocyst
/- heterozygous
C57BL/6 strain
wild-type allele
knockout allele
Blastocyst donor
129 strain
Blastocyst (3.5 days)
Foster mother
Chimeric mice
8
Germline transmission

-


Brown/Brown
black/black


-

or
Brown/black
Brown/black
Heterozygous germline knockout mice
9
homozygous knockout mouse
Knockout construct
Transfection/screening
/-
/-
injection
Chimeric mice
-/-
/-
/
25
25
50
Heterozygous mice
phenotype?
littermate control
Homozygous mice
10
The Function of AID
AID is expressed in activated B cells in germinal
center.
Spleen RNA
Mouse
Immunize with SRBC
In situ hybridization of spleen
Northern analysis of spleen RNA
unimmunized
immunized
AP-conjugated anti-digoxigenin Ab
PNA- FITC
Digoxigenin- probe
GC
AID mRNA
11
Knockout of AID to determine its function
Genomic map
Southern analysis
wild-type allele
targeted allele
mRNA
12
IgG and IgA deficiency in serum
Immunize mouse with SRBC
ELISA analysis of mouse serum
ELISA analysis of antibodies Against SRBC.
10 week-old mouse
/-
-/-
13
Lack of class switching in vitro
LPS
Day 7
mouse
spleen
ELISA analysis of culture supernatant
LPS IL4
LPS TGF-b
14
Somatic hypermutation defect
Immunized with NP-CGG
(CGG chicken g-globulin)
ELISA analysis of anti-NP antibody
Sequence analysis of IgH V region.
R replacement mutation
S silent mutation
15
Formation of large germinal centers
-/-
/-
Spleen section from mice immunized with SRBC
Anti-IgM-Texas Red
FITC-PNA (peanut agglutinin)
Immunofluorescence
16
Hyperplasia of lymphoid follicles
intestine
20-week
Isolated lymphoid follicles
Peyers patch
9 days
21 days
20 weeks
Hematoxylin/eosin stainnig
17
Hyper IgM (HIGM) syndrome
HIGM I CD40L deficiency
HIGM II no mutation in CD40L
18
HIMG II B cells do not undergo class switching in
vitro.
19
Somatic hypermutation defect
20
Large germinal center in HIGM II patients
Lymph node
Hematoxylin/eosin staining of tissue section
21
HIGM II patients have mutations in AID gene.
Disease linked with a region on chromosome 12
(12p13)
This region contains AID gene.