Fetal liver culture

1
Fetal liver culture
Knockout of DNA ligase IV
DNA ligase IV may be involved in V(D)J
recombination.
V
J
RAG1/2
V
J
DNA-PK, Artemis
V
J
XRCC-4 / DNA ligase IV
V
J
signal joint
coding joint
2
The knockout mice are embryonic lethal.
Day 13.5-14.5
Fetal liver (day 15-17.5)
Stromal cell (IL7)
B cells
DNA ligase IV is required for B cell maturation.
3
Adoptive Transfer
Knockout of DNA polymerase b
3
5
3
5
Base modification (e.g. methylation)
Modified base
3
5
3
5
Base excision repair
Modified base
3
5
3
5
DNA polymerase b
XRCC1 / DNA ligase III
3
5
3
5
4
Is DNA polymerase b involved in somatic
hypermutation?
Pol b knockout mice die around birth (perinatal).
5
Transfer of fetal liver cells
DNA pol b knockout embryo (day 18.5)
Recipient mouse (129/Sv, IgHa allotype)
(C57BL/6, IgHb allotype)
Lethal irradiation (g-ray)
Hematopoietic system depleted
Fetal liver cells
DNA pol b knockout B and T cells
6
Reconstitution of T and B cells
/-
-/-
7
Normal B cell response to immunization
Reconstituted mice
Immunize with NP-CGG
Anti-NP IgG levels Mutation frequency
DNA pol b is not required for somatic
hypermutation.
8
RAG-2-/- blastocyst complementation
ES cell
RAG2-/- mice
injection
RAG2-/- blastocyst
/-
Chimeric mice
Knockout first allele
HSC (RAG2-/-, X/)
HSC (RAG2/, X-/-)
-/-
B, T cells
B, T cells
Knockout second allele
(RAG2/, X-/-)
All the B and T cells in chimera are Knockout
cells.
9
Knockout of XBP-1 (X-box binding protein-1)
High expression levels in plasma cells.
Northern analysis of mRNA level in activated B
cells.
XBP-1 is expressed in other cell types.
XBP-1 is essential for hepatocytes.
XBP-1 knockout mice die during embryonic
development.
10
Reconstitution of B and T cells by
RAG2-/- blastocyst complementation
ES cell
XBP-1/-
XBP-1-/-
RAG2-/- blastocyst
Chimeric mice with XBP-/- B and T cells
11
Defect in immunoglobulin production
Serum Ig levels measured by ELISA
Spleen B cells stimulated with LPS.
Ig levels in culture supernatant measured by
ELISA
12
Defect in Ig secretion rather than class switching
Normal cell proliferation in response to
anti-CD40 antibody and IL-4.
Normal class switching determined by surface
expression of IgG
B cell
class switching (activated B cells)
Ig secretion (plasma cells)
13
Defective response to immunization
TNP-Ficoll T-independent antigen
TNP-CGG T-dependent antigen
Polyoma virus
Impaired survival
14
Conditional knockout
Deletion of gene in particular cell type and
developmental stage.
Cre-loxP system (bacterial phage P1)
loxP
loxP
34 bp
34 bp
Cre recombinase
15
Introduce loxP sites into gene of interest
wild-type
gene
/
ES cell
Wild-type
gene
Wild-type
gene
/flox
loxP
loxP
ES cell with floxed gene
Flox
gene
Floxed
16
Breed with cre-mice to delete floxed-gene
Gene to analyze
Cre-mouse
Floxed-mouse
flox
flox




CD19
CD19-cre

CD19
B cell-specific expression of cre
cre
CD19 promoter
flox
flox
CD19-cre
gene

CD19
Gene knockout in B cells
17
Generation of cre-mice
Knock-in
CD19 promoter
genome
CD19 gene
Homologous recombination
neo
tk
cre
Knock-in
CD19 promoter
genome
neo
cre
Express cre in B cells
18
Transgenic mice
promoter
Transgenic construct
cre
DNA randomly integrated into genome
19
Different strains of cre-mice
CD21-cre
CD19-cre
Immature B
Mature B
pre-B
pro-B
HSC
CLP
T cell
thymocyte
Lck-cre
IFN-a,b
Mx-cre
Poly(I)/poly(C) (dsRNA)
20
B cell-specific knockout of Blimp-1 (B lymphocyte
induced maturation protein-1)
Transcription factor
Expression induced during B cell differentiation
into plasma cells
Expressed in many cell lineages during embryonic
development
Knockout die during early embryonic stage.
CD19-cre is used to delete Blimp-1 in B cells.
Southern analysis of B cell DNA
Western analysis of B cell protein
21
Defective secretion of immunoglobulin
Immunization with NP-Ficoll (TI-antigen)
ELISA analysis of anti-NP antibody
-/-
control
Immunization with NP-KLH (T-dependent antigen)
22
ELISPOT assay
Determine the frequency of cells secreting a
cytokine or antibody.
Before activation
Frequency of T cells secreting IFN-g after
activation.
23
Reduction in the frequency of IgM secreting cells
Ig secreting
NP-Ficoll
Ig
control
Ag (NP) coated plate
-/-
AP-anti-Ig antibody
NP-KLH
color reaction
Ag (NP) coated plate
24
Defective production of secretive form of
Immunoglobulin
Western analysis of IgM from B cells activated
by LPS
Blimp-1 is required for plasma cell
differentiation.
25
Relevant part in book
Mice technique page 541-545
ELISPOT page 150