小鼠基因剔除技术（ knockout）
? 小鼠胚胎干细胞
– 体外传代
– 发育全能性（特别是能发育为生殖细胞）
? DNA同源重组技术
? 基因捕获技术（随机方法）
? 化学诱变技术（ ENU）
– ES细胞
– 小鼠个体
小鼠基因剔除技术的里程碑工作
? 1981Evans and Kaufman:（ Nature,1981 Jul 9;292(5819):154-6,Proc Natl Acad Sci
U S A,1981 Dec;78(12):7634-8）建立了 ES细胞
? 1986－ 1987 Robertson et al, Hooper et al，
Kuehn et al，：对 ES细胞进行遗传操作，并获得
ES细胞来源的小鼠个体（ Nature,1986 Oct 2-8;323(6087):445-8,Nature 1987 Mar
19-25;326(6110):292-5,Nature,1987 Mar 19-25;326(6110):295-8.）
? 1988 Thomas et al.,：在 ES细胞中建立基于同源重
组的基因打靶技术（ Cell,1987 Nov 6;51(3):503-12,）
? 1989 Jaenisch et al,建立 b2-microglobulin-
deficient基因剔除小鼠 (Nature,1989 Nov 23;342(6248):435-8.)
? 1994 Gu et al,将 Cre-LoxP系统用于 ES细胞的打靶，
建立条件性基因剔除技术（ Science,1994, 265:103,）
基因打靶 (Gene targeting)
? 基因剔除（常规、条件性）
(Gene Knock-out,conventional and conditional)
? 基因敲入 (Gene Knock-in)
? 基因捕获 (Gene Trap)
Early mouse development
From
Sedivy &
Joyner
“Gene
Targeting”
1992
Blastocyst 3.5 days
Inner Cell Mass
(128 cell stage)
1-totipotent
2-tissue culture
3-Transfectable
4-Selection
5- Differentiation
In vitro
Harvest blastocysts on day 3.5 by
flushing from the uterine lumem with
M2 medium
Plate out blastocysts in individual 10 mm dishes
on mitotically inactive MEF or STO feeder cells to
provide LIF and other factors
After about 96-120 hours in culture (C,D),the ICM can be
dislodged from the TE layer,washed and transplanted to
microdrops of medium containing trypsin to disperse the
clumps
After 1-2 days,blastocysts hatch (A)
and attach to the dish by migration of
the trophectoderm (TE),while the
inner cell mass (ICM) grows (B)
Transfer disaggregated contents to a fresh feeder cell
tissue culture well and inspect daily for signs of
differentiation,Primary cell colonies are clearly visible as
distinct clumps
Generating ES Cells
~30 cells
NM-2细胞的核型图
Stem cell cultures
Mouse Embryonic Fibroblast Cells:
Mitotically inactivated by irradiation
or mitomycin C
ES cells,Medium with 15% FCS
1000u/ml LIF
CO2, 7-10%
LIF (leukaemia inhibitory factor),
Maintains stem cells in an undifferentiated state
ES Cells Differentiation
Tissues
Muscle
Cells
Drug or Growth factors
Bone
Cells
Blood
Cells
Blood
Vessels
Neuronal
Development
Gene targeting in embryonic stem cells
? Targeting vector with the desired change is
electroporated into ES cells
? Homologous recombinants are identified by drug
selection &molecular screening
? Recombinant ES clones are injected into blastocysts
to produce chimeric mice
? Chimeras are bred to produce heterozygotes
? Heterozygotes are intercrossed to produce
homozygous mutants
打靶载体的设计
? 选用同源 DNA
? 同源臂的长度
? 正、负选择
Factors influencing targeting efficiency
? isogenic DNA (perfect homology)
10-25 fold
van Deursen J,Wieringa B,Targeting of the creatine kinase M gene in embryonic stem cells using
isogenic and nonisogenic vectors,Nucleic Acids Res,20:3815-20,1992.
? size of region of homology
exponential relationship
Capecchi MR,Altering the genome by homologous recombination.
Science,244:1288-9,1989
? robust screen!
Positive controls
Homologous recombination
1- Length of homologous sequences
Hom,Rec,Efficiency
Base pair
25bp 2000bp
2- Isogenic DNA
NEOMYCIN
X X
Gene targeting
NEOMYCIN
Total 4 Kbp (each arm not less than 500bp)
Delete coding sequences
Change reading frame
Transcription of Neo in antisense direction
Positive selection:
- neomycin phosphotransferase (neo)
- hygromycin phosphotransferase (hyg)
- puromycine (pur)
- hypoxanthine phosphoribosyltransferase (hprt)
Negative selection
- thymidine kinase (tk)
- cytosine deaminase (cd)
- hypoxanthine phosphoribosyltransferase (hprt)
SELECTION MARKER GENES
Positive selection:
ES cells that have the targeting vector,neo resistant
Negative selection:
Select against ES cells with random vector integration
sensitive to anti-herpes drugs,FIAU,ganciclovir
Molecular screen:
Eliminates random integrants without HSVtk
Verifies recombination on both ends
3 HSVtk1 neo
31 neo5’probe
31 2
targeted allele
endogenous allele
random
3’probe
3 H1 neo
random
HR1 1 2 HR23
HR1 b-geo HR23
HR1 b-geo HR23
Genomic
Locus
Targeting
Vector
Mutant
Locus
HSVtk
STOP
CODONS
Positive-Negative Selection
S c r e e n i n g P l a t e - G r o w f o r
D N A,r e s t r i c t i o n d i g e s t,
t h e n S o u t h e r n
M a s t e r P l a t e - F r e e z e c e l l s
o r c o n t i n u e t o g r o w a n d
s u b p l a t e
P i c k c l o n e s
a n d t r y p s i n i z e
p l a t e a n d
g r o w
Screening ES Cell Clones
HR1 1 2 HR23
HR1 b-geo HR23
HR1 b-geo HR23
Genomic
Locus
Targeting
Vector
Mutant
Locus
HSVtk
1 2
3
PCR SCREENING:
1-2 = WT locus
1-3 = MT locus
A
A
A A
A
A C
CC
C
C
B
5’ 3’
SOUTHERN BLOT SCREENING:
A-A,C-C = both WT and MT,but different
sizes,when probed with 5’ and 3’
probes,respectively
A-B,B-C = Specific to MT when probed
with the same probes
Strategies for Screening
Typically:
Homologous recombination accounts for < 2% of all integration
events.
1 in 10 million cells will undergo homologous recombination.
1 in 200 cells that come through positive-negative selection will
turn out to be correctly targeted.
Low Homologous Recombination Possibility
Timeline,generation of ES cell-derived mice
Introduce
targeting
vector into
ES cells
Identify
homologous
recombinants
by DNA
analysis
Identify mouse
Chimeras with
high ES cell
contribution Germline transmission
Begin
analysis
0 2 4 8 10 126
Drug
selection
Colony growth
and expansion
Inject
clones
into
blastocysts
Sexual
maturation
of chimeras
Identify
male and
female
heterozygotes
Sexual
maturation of
heterozygotes
Identify
homozygotes
How do you get -/- ES cells?
?Select for gene conversion by increasing G418
?Retarget locus with different selectable marker
?(Derive ES cells from homozygous mutants)
difficult!
alternative,embryonic fibroblasts from -/- animal
ICM
Normal
C57BL/6
Blastocyst
(black)
ES cells
129/SvJ
(agouti)
agouti black
Breeding Chimeras (knock-out
founder)
? Chimera - the founder
– germ-line transmission - usually the ES cells
are derived from a 129 strain (agouti or white
colour) and the ES cells are injected into a
C57Bl/6 blastocyst (black),The more that the
ES cells contribute to the genome of the mouse,
the more the coat colour will be agouti,The
chimera mouse is usually,tiger” striped.
Breeding Chimeras (knock-out
founder)cont
? Males that are 40% to 100% based on
agouti coat colour should be bred
? Females should not be bred (low incidence
of success) ES cells are male.
? Breed aggressively- rotate females through
male's cage,If the male produces more than
6 litters without transmitting,not likely to
go germline and should be sac'ed
X
X
1 WT 2 Hetero 1 Homo
基因打靶技术的局限性
? 胚胎致死
? 所有细胞都改变
? 功能代偿
? 冗余
? 单元与系统的关系
条件性打靶技术
conditional knockout
? Cre/loxP recombinase
? FLP/FRT recombinase
? Inducible system
Gu,H.et al,Deletion of a DNA polymerase B gene segment
in T cells using cell-type specific gene targeting,
1994,Science,265:103,
Hua GU Van Klaus Rajewsky
Cre-lox technology
Cre – a site-specific recombinase enzyme from the P1 phage.
Recognises a 34bp DNA sequence loxP =
Cre
Cre
Cre
Principles
AND BREED THE TWO TOGETHER - gene is knocked out only
in tissues where Cre is expressed.
Need to create and maintain two strains (lines) of mice
1) A line of mice in which loxP sites have been inserted around
the gene of interest.
GENE
2) A line of mice that express Cre recombinase from a tissue-
specific promoter (e.g,directs Cre expression to pancreas only).
Cre
Conditional targeting by homologous
recombination in ES cells
ATG
= regions of homologous DNA sequence
Endogenous gene
Targeting vector
neoRATG TK
neoR
Electroporate target vector into ES cells and perform positive-
negative selection,PCR and southern blotting to isolate clones that
have integrated correctly,so that genomic DNA of ES cells =
5
Transiently transfect these ES cells with constitutive Cre construct,
creating cells that are...
5
5neoR
Select and isolate clones of cells that are now G418-sensitive
or
or5
neo
5
ES cells that whose genomic DNA is modified as above can be
used to create a conventional knockout,
5
ES cells containing the ?floxed? allele form the basis of the
tissue- specific knockout,Inject into blastocysts to get chimeras,
breed from chimeras to get heterozygotes and breed
heterozygotes together to get homozygous floxed mice,These
can be bred with the appropriate Cre transgenic line.
ATG
neoR
Alternative Floxing strategy - avoids extra ES cell step
Endogenous gene
Targeting vector
loxP site
FRT site
TK
neoR
Electroporate targeting vector into ES cells and perform selection
as before
Breed these mice with a line that constitutively expresses Flp
recombinase from the b-actin promoter.
This removes the neoR cassette and gives the floxed allele
Inject into blastocysts,create chimeras and breed these to get
heterozygotes as before.
Common Used Cre-transgenic Mouse Lines
WWW.mshri.on.ca/nagy/cre-pub.html
Promoter used Tissue
CMV Germline
Nestin Neuronal lineage
Lck T cells
CD19 B cells
K14 Keratinocytes
CMV-ERT Tamoxifen inducible
Mx1 IFN inducible
Resume,knock out gene in all tissues - mouse dies
x
Create floxed mice Create hemizygous Cre mice
flox/flox
Cross mice for a couple of generations to get Cre+
flox/flox homozygotes
flox/flox
Temporal induction of Cre expression,2
Tamoxifen-inducible system
Uses a fusion protein combining activity of Cre and a mutant form
of the ligand binding domain of the estrogen receptor (ERTM)
Cre-ERTM does not bind estrogen,but does bind 4-OH-tamoxifen
Hsp90
ERTM
Cre
cell
TM
Upon addition of tamoxifen,
Cre-ERTM is released from
Hsp90,allowing access to
the nucleus and Cre-
mediated recombination
flox/flox
Cre-ERTM
Create floxed/Cre double transgenics as before
Cre is being expressed in tissue of interest (e.g,pancreas),but is
sequestered in cytoplasm and inactive
Temporal induction of Cre expression,2
flox/flox
Cre-ERTM
Inject with tamoxifen
Cre is released into nucleus,leading to knockout of gene only in
tissues that are exposed to tamoxifen and where Cre is being
expressed.
Oviduct
The recipient female is mated
with a vasectomised male,
Over the next 2 days,her
uterine wall swells and
vascularises,ready for
implantation of blastocysts
Uterine transfer of
chimeric blastocysts
The female gives birth to a litter of
variably chimeric mice
基因敲入 (Gene Knock-in)
?基因的时空性表达
?基因或基因元件的突变研究
?报告基因,b- galactosidase,Luciferase,GFP,others…,
-
X X
Neo
Knock-in Strategy
Wt chromosome
KI construct
Neo
Homologous recombination
Cre mediated recombination
Recombinated chromosome
loxP
Mutated chromosme
基因捕获 (Gene Trap)
? 增强子 捕获
? 基因捕获
? 启动子捕获
? Poly A捕获
? 启动子和 Poly A捕获
Gene traps in ES cells
? Insertional mutagenesis
? Introduction of gene trap vectors into
ES cells (electroporation or retrovirus)
? Screen ES cell clones
? Generate mice for phenotypic analysis
GENE TRAPPING USING EXPRESSION-DEFECTIVE TRANSGENES
1,promoter-less reporter gene for gene trapping
2,pA-less selection marker for polyA-addition trapping
P,promoter; E1-E4,exons1-4; pA,polyadenylation signal; SA,splice acceptor sequence,SD,splice donor sequence;
INSERTIONAL MUTAGENESIS IN MICE,NEW PERSPECTIVES AND TOOLS,
Nature Reviews Genetics 6,568-580 (2005)
Inverse PCR
Used to obtain (clone) sequence adjacent to ONE known sequence
Standard PCR requires 2 regions of known sequence
Digest target DNA with restriction enzyme and ligate into circles
Use 2 outward pointing primers to amplify around the circle towards each other
RE RE RERE RE
RE
RE
known sequence
known sequence
known sequence known sequence
3’ -RACE
5’ -GACTCGAGTCGACATCGA(T)17-3’
Xho I Sal I Cla I Oligo(dT) adaptor primer
AAAAAAAAAAATCGATGTCGACTCGAGTC-3’
TTTTTTTTTTTAGCTACAGCTGAGCTCAG-5’
reverse transcription
PCR
AAAAAAAAAATCGATGTCGACTCGAGTC-3’
TTTTTTTTTTAGCTACAGCTGAGCTCAG-5’
GSP
adaptor primer
AAAAAAAAAA-3’
TTTTTTTTTTTAGCTACAGCTGAGCTCAG-5’
mRNA
RE site
RE siteRE site
digest with restriction enzymes and clone
GSP1
5’ -RACE
AAAAAAAAAA-3’
1,reverse transcription
3,Anneal primers
and do PCR
mRNA
RE site RE site
digest with restriction enzymes and clone
5’ -cap
remove RNA and GSP1
CCCCC
2,terminal transferase
+ dCTP
3’ -CCCCC
5’ -GACTCGAGTCGACATCGA(G)17-3’
Xho I Sal I Cla I
5’ -GACTCGAGTCGACATCGAGGGGG-3’
GSP2
CCCCC
5’ -GACTCGAGTCGACATCGAGGGGG
adaptor primer
Nature Genetics (2001) 28,198-200
Insertion outside
coding sequence
Gene lacking
secretory signal
Gene with upstream
secretory signal
Bone fide secretory
proteins
http://www.genetrap.org
Secretory
Gene Traps
Comparison of genetically engineered mice
created by pronuclear injection & ES derived mice
TRANSGENIC ES DERIVED
Genetic action dominant Recessive or dominant
Insertion site random Targeted to endogenous
Copy number Variable,
1 to >100
Heterozygotes 1,
homozygotes 2
expression Usually integration
site dependent
Usually same as
endogenous
time 6-9 months 1.5 -2 years
cost >$3000 >$12,000
other Insertional mutation Effects on neighboring
genes
IgM and TCR Structure
Rearranged TcR b?1° transcript
Spliced TcR b?mRNA
L & Vb
x52 Db1 J Cb1 Db2 J Cb2
Germline TcR b
TcR b gene rearrangement
SOMATIC RECOMBINATION
D-J Joining
V-DJ joining
C-VDJ joining
Alternative D region usage
V,D,J flanking sequences
Va 7 23 9
Sequencing upstream and downstream of V,D and J elements revealed
conserved sequences of 7,23,9 and 12 nucleotides.
Ja7129
Db7129 7 12 9
Vb 7 23 9 Jb7239
Rearrangement detected by PCR and hybridization
1 10 100 1 0 00 1 0 00 0
P E -A, CD 4 P E - A
1
10
100
1 0 00
1 0 00 0
P
e
r
CP
-Cy
5
-
5-A
,CD
8
P
e
rCP
-Cy
5-5
-A
3.1 84.8
2 10.1
CD4
WT Thymus
1 10 100 1 0 00 1 0 00 0
F IT C- A, CD 2 5 F IT C- A
1
10
100
1 0 00
1 0 00 0
P
E
-A
,CD
44
P
E
-A
1 10 100 1 0 00 1 0 00 0
F IT C- A, CD 2 5 F IT C- A
1
10
100
1 0 00
1 0 00 0
P
E
-A
,CD
44
P
E
-A
11.3 7.6
56.8 24.4
CD25
WT
CD4-CD8-
DN
CD8+
CD4+
CD4+CD8+
DP
CD44-
CD25-
CD44+
DN1
CD25+
DN3
CD44+CD25+
DN2
L & Vb
x52 Db1 J Cb1 Db2 J Cb2
Germline TcR b
Eb? Vb14
L & Vb
x52 Db1 J Cb1 Db2 J Cb2
Germline TcR b
loxP Vb14
Deletion of the mouse T-cell receptor b?gene enhancer
blocks ab T-cell Development
L & Vb
x52 Db1 J Cb1 Db2 J Cb2
Germline TcR b
Eb? Vb14
L & Vb
x52 Db1 J Cb1 Db2 J Cb2
Germline TcR b
Eb? Vb14
The role of CIN85 in lymphocyte development
and activation
cd2ap
Critical for stabilizing contacts between T cells and APC
cells by driving CD2 clustering,and cytoskeletal
polarization.
cd2ap-/- mice
Immune function compromised,but the mice dies at 6 to 7
weeks of age from renal failure.
PRO CCSH3 SH3 SH3
N N N
1kb Probe
N loxP N
neor
N
5 kb
3.3 kb
X chromoseome
Construct Making
N loxP N
neor
XhoI Sal I
XhoI
XhoI Sal I Hind III
SacI XhoI
Construct Making
pLZ-neo R
4.4kb
pGEM-30
3.0kb
Xba I Xho I loxP Sal I
HindIII
SacI
HindIII
SacI
Backbone plasmids for conditional KO
ScaI ScaI
N loxP N
neor
Xba I Xho I Sal I
neorXho I Sac I
Cut with
Xba I /Sal I/ Sca I
Cut with
Xho I /Sac I/ Sca I
Vector cut with Xba I and Sac I
Three molecules ligation
Xho I
Construct Making
loxP
neor
Construct linearized with Xho I
10-20 x 106 ES cells transfected 30-50mg
vector
XhoI site XhoI site
PRO CCSH3 SH3 SH3CIN85
wild-type
targeted
floxed
deleted
N N N
1kb Probe
N loxP N
neor
N
N N
N N
5 kb
3.3 kb
6.8 kb
4.8 kb
2R
C9 C10
2R
C10
CIN85- CIN85+
5 kb
3.3kb
X chromoseome
CIN85 flox
WT CIN85-
CD4
TCR b
1 10 100 1 0 00 1 0 00 0
P e rCP - C y 5 - 5- A
1
10
100
1 0 00
1 0 00 0
A
P
C-A
4,3 8 7 7,8
1 4,63,2 6
1 10 100 1 0 00 1 0 00 0
P e rCP - C y 5 - 5- A
1
10
100
1 0 00
1 0 00 0
A
P
C-A
3,1 7 8 1,6
1 2,23,0 4
1 10 100 1 0 00 1 0 00 0
F IT C- A
1
10
100
1 0 00
1 0 00 0
P
E
-A
0,2 3 0,0 1 3
2 5,57 4,2
1 10 100 1 0 00 1 0 00 0
F IT C- A
1
10
100
1 0 00
1 0 00 0
P
E
-A
0,1 9 0,0 1 1
2 0,47 9,4
1 10 100 1 0 00 1 0 00 0
F IT C- A, c f s e F I T C- A
0
20
40
60
80
100
%
o
f
M
a
x
1 10 100 1 0 00 1 0 00 0
F IT C- A, c f s e F I T C- A
0
20
40
60
80
100
%
o
f
M
a
x
1 10 100 1 0 00 1 0 00 0
F IT C- A, c f s e F I T C- A
0
20
40
60
80
100
%
o
f
M
a
x
1 10 100 1 0 00 1 0 00 0
F IT C- A, c f s e F I T C- A
0
20
40
60
80
100
%
o
f
M
a
x
WT
CIN85-
4.5 day CFSE
CD4 CD8
CIN85是 T 细胞受体信号
通路的调控蛋白。用荧光
染料 CFSE标记从 CIN85
基因剔除小鼠和野生型小
鼠的脾脏中分离 T淋巴细
胞，并用 5 mg/ml CD3进
行刺激,4.5天后，用流
式细胞仪分析 T细胞受体
介导的细胞增殖。细胞内
CFSE的荧光强度随细胞
分裂而减弱，结果显示
CIN85基因剔除的 CD4和
CD8 T细胞分裂得更快。
non-sti
sti
谢谢！
? 小鼠胚胎干细胞
– 体外传代
– 发育全能性（特别是能发育为生殖细胞）
? DNA同源重组技术
? 基因捕获技术（随机方法）
? 化学诱变技术（ ENU）
– ES细胞
– 小鼠个体
小鼠基因剔除技术的里程碑工作
? 1981Evans and Kaufman:（ Nature,1981 Jul 9;292(5819):154-6,Proc Natl Acad Sci
U S A,1981 Dec;78(12):7634-8）建立了 ES细胞
? 1986－ 1987 Robertson et al, Hooper et al，
Kuehn et al，：对 ES细胞进行遗传操作，并获得
ES细胞来源的小鼠个体（ Nature,1986 Oct 2-8;323(6087):445-8,Nature 1987 Mar
19-25;326(6110):292-5,Nature,1987 Mar 19-25;326(6110):295-8.）
? 1988 Thomas et al.,：在 ES细胞中建立基于同源重
组的基因打靶技术（ Cell,1987 Nov 6;51(3):503-12,）
? 1989 Jaenisch et al,建立 b2-microglobulin-
deficient基因剔除小鼠 (Nature,1989 Nov 23;342(6248):435-8.)
? 1994 Gu et al,将 Cre-LoxP系统用于 ES细胞的打靶，
建立条件性基因剔除技术（ Science,1994, 265:103,）
基因打靶 (Gene targeting)
? 基因剔除（常规、条件性）
(Gene Knock-out,conventional and conditional)
? 基因敲入 (Gene Knock-in)
? 基因捕获 (Gene Trap)
Early mouse development
From
Sedivy &
Joyner
“Gene
Targeting”
1992
Blastocyst 3.5 days
Inner Cell Mass
(128 cell stage)
1-totipotent
2-tissue culture
3-Transfectable
4-Selection
5- Differentiation
In vitro
Harvest blastocysts on day 3.5 by
flushing from the uterine lumem with
M2 medium
Plate out blastocysts in individual 10 mm dishes
on mitotically inactive MEF or STO feeder cells to
provide LIF and other factors
After about 96-120 hours in culture (C,D),the ICM can be
dislodged from the TE layer,washed and transplanted to
microdrops of medium containing trypsin to disperse the
clumps
After 1-2 days,blastocysts hatch (A)
and attach to the dish by migration of
the trophectoderm (TE),while the
inner cell mass (ICM) grows (B)
Transfer disaggregated contents to a fresh feeder cell
tissue culture well and inspect daily for signs of
differentiation,Primary cell colonies are clearly visible as
distinct clumps
Generating ES Cells
~30 cells
NM-2细胞的核型图
Stem cell cultures
Mouse Embryonic Fibroblast Cells:
Mitotically inactivated by irradiation
or mitomycin C
ES cells,Medium with 15% FCS
1000u/ml LIF
CO2, 7-10%
LIF (leukaemia inhibitory factor),
Maintains stem cells in an undifferentiated state
ES Cells Differentiation
Tissues
Muscle
Cells
Drug or Growth factors
Bone
Cells
Blood
Cells
Blood
Vessels
Neuronal
Development
Gene targeting in embryonic stem cells
? Targeting vector with the desired change is
electroporated into ES cells
? Homologous recombinants are identified by drug
selection &molecular screening
? Recombinant ES clones are injected into blastocysts
to produce chimeric mice
? Chimeras are bred to produce heterozygotes
? Heterozygotes are intercrossed to produce
homozygous mutants
打靶载体的设计
? 选用同源 DNA
? 同源臂的长度
? 正、负选择
Factors influencing targeting efficiency
? isogenic DNA (perfect homology)
10-25 fold
van Deursen J,Wieringa B,Targeting of the creatine kinase M gene in embryonic stem cells using
isogenic and nonisogenic vectors,Nucleic Acids Res,20:3815-20,1992.
? size of region of homology
exponential relationship
Capecchi MR,Altering the genome by homologous recombination.
Science,244:1288-9,1989
? robust screen!
Positive controls
Homologous recombination
1- Length of homologous sequences
Hom,Rec,Efficiency
Base pair
25bp 2000bp
2- Isogenic DNA
NEOMYCIN
X X
Gene targeting
NEOMYCIN
Total 4 Kbp (each arm not less than 500bp)
Delete coding sequences
Change reading frame
Transcription of Neo in antisense direction
Positive selection:
- neomycin phosphotransferase (neo)
- hygromycin phosphotransferase (hyg)
- puromycine (pur)
- hypoxanthine phosphoribosyltransferase (hprt)
Negative selection
- thymidine kinase (tk)
- cytosine deaminase (cd)
- hypoxanthine phosphoribosyltransferase (hprt)
SELECTION MARKER GENES
Positive selection:
ES cells that have the targeting vector,neo resistant
Negative selection:
Select against ES cells with random vector integration
sensitive to anti-herpes drugs,FIAU,ganciclovir
Molecular screen:
Eliminates random integrants without HSVtk
Verifies recombination on both ends
3 HSVtk1 neo
31 neo5’probe
31 2
targeted allele
endogenous allele
random
3’probe
3 H1 neo
random
HR1 1 2 HR23
HR1 b-geo HR23
HR1 b-geo HR23
Genomic
Locus
Targeting
Vector
Mutant
Locus
HSVtk
STOP
CODONS
Positive-Negative Selection
S c r e e n i n g P l a t e - G r o w f o r
D N A,r e s t r i c t i o n d i g e s t,
t h e n S o u t h e r n
M a s t e r P l a t e - F r e e z e c e l l s
o r c o n t i n u e t o g r o w a n d
s u b p l a t e
P i c k c l o n e s
a n d t r y p s i n i z e
p l a t e a n d
g r o w
Screening ES Cell Clones
HR1 1 2 HR23
HR1 b-geo HR23
HR1 b-geo HR23
Genomic
Locus
Targeting
Vector
Mutant
Locus
HSVtk
1 2
3
PCR SCREENING:
1-2 = WT locus
1-3 = MT locus
A
A
A A
A
A C
CC
C
C
B
5’ 3’
SOUTHERN BLOT SCREENING:
A-A,C-C = both WT and MT,but different
sizes,when probed with 5’ and 3’
probes,respectively
A-B,B-C = Specific to MT when probed
with the same probes
Strategies for Screening
Typically:
Homologous recombination accounts for < 2% of all integration
events.
1 in 10 million cells will undergo homologous recombination.
1 in 200 cells that come through positive-negative selection will
turn out to be correctly targeted.
Low Homologous Recombination Possibility
Timeline,generation of ES cell-derived mice
Introduce
targeting
vector into
ES cells
Identify
homologous
recombinants
by DNA
analysis
Identify mouse
Chimeras with
high ES cell
contribution Germline transmission
Begin
analysis
0 2 4 8 10 126
Drug
selection
Colony growth
and expansion
Inject
clones
into
blastocysts
Sexual
maturation
of chimeras
Identify
male and
female
heterozygotes
Sexual
maturation of
heterozygotes
Identify
homozygotes
How do you get -/- ES cells?
?Select for gene conversion by increasing G418
?Retarget locus with different selectable marker
?(Derive ES cells from homozygous mutants)
difficult!
alternative,embryonic fibroblasts from -/- animal
ICM
Normal
C57BL/6
Blastocyst
(black)
ES cells
129/SvJ
(agouti)
agouti black
Breeding Chimeras (knock-out
founder)
? Chimera - the founder
– germ-line transmission - usually the ES cells
are derived from a 129 strain (agouti or white
colour) and the ES cells are injected into a
C57Bl/6 blastocyst (black),The more that the
ES cells contribute to the genome of the mouse,
the more the coat colour will be agouti,The
chimera mouse is usually,tiger” striped.
Breeding Chimeras (knock-out
founder)cont
? Males that are 40% to 100% based on
agouti coat colour should be bred
? Females should not be bred (low incidence
of success) ES cells are male.
? Breed aggressively- rotate females through
male's cage,If the male produces more than
6 litters without transmitting,not likely to
go germline and should be sac'ed
X
X
1 WT 2 Hetero 1 Homo
基因打靶技术的局限性
? 胚胎致死
? 所有细胞都改变
? 功能代偿
? 冗余
? 单元与系统的关系
条件性打靶技术
conditional knockout
? Cre/loxP recombinase
? FLP/FRT recombinase
? Inducible system
Gu,H.et al,Deletion of a DNA polymerase B gene segment
in T cells using cell-type specific gene targeting,
1994,Science,265:103,
Hua GU Van Klaus Rajewsky
Cre-lox technology
Cre – a site-specific recombinase enzyme from the P1 phage.
Recognises a 34bp DNA sequence loxP =
Cre
Cre
Cre
Principles
AND BREED THE TWO TOGETHER - gene is knocked out only
in tissues where Cre is expressed.
Need to create and maintain two strains (lines) of mice
1) A line of mice in which loxP sites have been inserted around
the gene of interest.
GENE
2) A line of mice that express Cre recombinase from a tissue-
specific promoter (e.g,directs Cre expression to pancreas only).
Cre
Conditional targeting by homologous
recombination in ES cells
ATG
= regions of homologous DNA sequence
Endogenous gene
Targeting vector
neoRATG TK
neoR
Electroporate target vector into ES cells and perform positive-
negative selection,PCR and southern blotting to isolate clones that
have integrated correctly,so that genomic DNA of ES cells =
5
Transiently transfect these ES cells with constitutive Cre construct,
creating cells that are...
5
5neoR
Select and isolate clones of cells that are now G418-sensitive
or
or5
neo
5
ES cells that whose genomic DNA is modified as above can be
used to create a conventional knockout,
5
ES cells containing the ?floxed? allele form the basis of the
tissue- specific knockout,Inject into blastocysts to get chimeras,
breed from chimeras to get heterozygotes and breed
heterozygotes together to get homozygous floxed mice,These
can be bred with the appropriate Cre transgenic line.
ATG
neoR
Alternative Floxing strategy - avoids extra ES cell step
Endogenous gene
Targeting vector
loxP site
FRT site
TK
neoR
Electroporate targeting vector into ES cells and perform selection
as before
Breed these mice with a line that constitutively expresses Flp
recombinase from the b-actin promoter.
This removes the neoR cassette and gives the floxed allele
Inject into blastocysts,create chimeras and breed these to get
heterozygotes as before.
Common Used Cre-transgenic Mouse Lines
WWW.mshri.on.ca/nagy/cre-pub.html
Promoter used Tissue
CMV Germline
Nestin Neuronal lineage
Lck T cells
CD19 B cells
K14 Keratinocytes
CMV-ERT Tamoxifen inducible
Mx1 IFN inducible
Resume,knock out gene in all tissues - mouse dies
x
Create floxed mice Create hemizygous Cre mice
flox/flox
Cross mice for a couple of generations to get Cre+
flox/flox homozygotes
flox/flox
Temporal induction of Cre expression,2
Tamoxifen-inducible system
Uses a fusion protein combining activity of Cre and a mutant form
of the ligand binding domain of the estrogen receptor (ERTM)
Cre-ERTM does not bind estrogen,but does bind 4-OH-tamoxifen
Hsp90
ERTM
Cre
cell
TM
Upon addition of tamoxifen,
Cre-ERTM is released from
Hsp90,allowing access to
the nucleus and Cre-
mediated recombination
flox/flox
Cre-ERTM
Create floxed/Cre double transgenics as before
Cre is being expressed in tissue of interest (e.g,pancreas),but is
sequestered in cytoplasm and inactive
Temporal induction of Cre expression,2
flox/flox
Cre-ERTM
Inject with tamoxifen
Cre is released into nucleus,leading to knockout of gene only in
tissues that are exposed to tamoxifen and where Cre is being
expressed.
Oviduct
The recipient female is mated
with a vasectomised male,
Over the next 2 days,her
uterine wall swells and
vascularises,ready for
implantation of blastocysts
Uterine transfer of
chimeric blastocysts
The female gives birth to a litter of
variably chimeric mice
基因敲入 (Gene Knock-in)
?基因的时空性表达
?基因或基因元件的突变研究
?报告基因,b- galactosidase,Luciferase,GFP,others…,
-
X X
Neo
Knock-in Strategy
Wt chromosome
KI construct
Neo
Homologous recombination
Cre mediated recombination
Recombinated chromosome
loxP
Mutated chromosme
基因捕获 (Gene Trap)
? 增强子 捕获
? 基因捕获
? 启动子捕获
? Poly A捕获
? 启动子和 Poly A捕获
Gene traps in ES cells
? Insertional mutagenesis
? Introduction of gene trap vectors into
ES cells (electroporation or retrovirus)
? Screen ES cell clones
? Generate mice for phenotypic analysis
GENE TRAPPING USING EXPRESSION-DEFECTIVE TRANSGENES
1,promoter-less reporter gene for gene trapping
2,pA-less selection marker for polyA-addition trapping
P,promoter; E1-E4,exons1-4; pA,polyadenylation signal; SA,splice acceptor sequence,SD,splice donor sequence;
INSERTIONAL MUTAGENESIS IN MICE,NEW PERSPECTIVES AND TOOLS,
Nature Reviews Genetics 6,568-580 (2005)
Inverse PCR
Used to obtain (clone) sequence adjacent to ONE known sequence
Standard PCR requires 2 regions of known sequence
Digest target DNA with restriction enzyme and ligate into circles
Use 2 outward pointing primers to amplify around the circle towards each other
RE RE RERE RE
RE
RE
known sequence
known sequence
known sequence known sequence
3’ -RACE
5’ -GACTCGAGTCGACATCGA(T)17-3’
Xho I Sal I Cla I Oligo(dT) adaptor primer
AAAAAAAAAAATCGATGTCGACTCGAGTC-3’
TTTTTTTTTTTAGCTACAGCTGAGCTCAG-5’
reverse transcription
PCR
AAAAAAAAAATCGATGTCGACTCGAGTC-3’
TTTTTTTTTTAGCTACAGCTGAGCTCAG-5’
GSP
adaptor primer
AAAAAAAAAA-3’
TTTTTTTTTTTAGCTACAGCTGAGCTCAG-5’
mRNA
RE site
RE siteRE site
digest with restriction enzymes and clone
GSP1
5’ -RACE
AAAAAAAAAA-3’
1,reverse transcription
3,Anneal primers
and do PCR
mRNA
RE site RE site
digest with restriction enzymes and clone
5’ -cap
remove RNA and GSP1
CCCCC
2,terminal transferase
+ dCTP
3’ -CCCCC
5’ -GACTCGAGTCGACATCGA(G)17-3’
Xho I Sal I Cla I
5’ -GACTCGAGTCGACATCGAGGGGG-3’
GSP2
CCCCC
5’ -GACTCGAGTCGACATCGAGGGGG
adaptor primer
Nature Genetics (2001) 28,198-200
Insertion outside
coding sequence
Gene lacking
secretory signal
Gene with upstream
secretory signal
Bone fide secretory
proteins
http://www.genetrap.org
Secretory
Gene Traps
Comparison of genetically engineered mice
created by pronuclear injection & ES derived mice
TRANSGENIC ES DERIVED
Genetic action dominant Recessive or dominant
Insertion site random Targeted to endogenous
Copy number Variable,
1 to >100
Heterozygotes 1,
homozygotes 2
expression Usually integration
site dependent
Usually same as
endogenous
time 6-9 months 1.5 -2 years
cost >$3000 >$12,000
other Insertional mutation Effects on neighboring
genes
IgM and TCR Structure
Rearranged TcR b?1° transcript
Spliced TcR b?mRNA
L & Vb
x52 Db1 J Cb1 Db2 J Cb2
Germline TcR b
TcR b gene rearrangement
SOMATIC RECOMBINATION
D-J Joining
V-DJ joining
C-VDJ joining
Alternative D region usage
V,D,J flanking sequences
Va 7 23 9
Sequencing upstream and downstream of V,D and J elements revealed
conserved sequences of 7,23,9 and 12 nucleotides.
Ja7129
Db7129 7 12 9
Vb 7 23 9 Jb7239
Rearrangement detected by PCR and hybridization
1 10 100 1 0 00 1 0 00 0
P E -A, CD 4 P E - A
1
10
100
1 0 00
1 0 00 0
P
e
r
CP
-Cy
5
-
5-A
,CD
8
P
e
rCP
-Cy
5-5
-A
3.1 84.8
2 10.1
CD4
WT Thymus
1 10 100 1 0 00 1 0 00 0
F IT C- A, CD 2 5 F IT C- A
1
10
100
1 0 00
1 0 00 0
P
E
-A
,CD
44
P
E
-A
1 10 100 1 0 00 1 0 00 0
F IT C- A, CD 2 5 F IT C- A
1
10
100
1 0 00
1 0 00 0
P
E
-A
,CD
44
P
E
-A
11.3 7.6
56.8 24.4
CD25
WT
CD4-CD8-
DN
CD8+
CD4+
CD4+CD8+
DP
CD44-
CD25-
CD44+
DN1
CD25+
DN3
CD44+CD25+
DN2
L & Vb
x52 Db1 J Cb1 Db2 J Cb2
Germline TcR b
Eb? Vb14
L & Vb
x52 Db1 J Cb1 Db2 J Cb2
Germline TcR b
loxP Vb14
Deletion of the mouse T-cell receptor b?gene enhancer
blocks ab T-cell Development
L & Vb
x52 Db1 J Cb1 Db2 J Cb2
Germline TcR b
Eb? Vb14
L & Vb
x52 Db1 J Cb1 Db2 J Cb2
Germline TcR b
Eb? Vb14
The role of CIN85 in lymphocyte development
and activation
cd2ap
Critical for stabilizing contacts between T cells and APC
cells by driving CD2 clustering,and cytoskeletal
polarization.
cd2ap-/- mice
Immune function compromised,but the mice dies at 6 to 7
weeks of age from renal failure.
PRO CCSH3 SH3 SH3
N N N
1kb Probe
N loxP N
neor
N
5 kb
3.3 kb
X chromoseome
Construct Making
N loxP N
neor
XhoI Sal I
XhoI
XhoI Sal I Hind III
SacI XhoI
Construct Making
pLZ-neo R
4.4kb
pGEM-30
3.0kb
Xba I Xho I loxP Sal I
HindIII
SacI
HindIII
SacI
Backbone plasmids for conditional KO
ScaI ScaI
N loxP N
neor
Xba I Xho I Sal I
neorXho I Sac I
Cut with
Xba I /Sal I/ Sca I
Cut with
Xho I /Sac I/ Sca I
Vector cut with Xba I and Sac I
Three molecules ligation
Xho I
Construct Making
loxP
neor
Construct linearized with Xho I
10-20 x 106 ES cells transfected 30-50mg
vector
XhoI site XhoI site
PRO CCSH3 SH3 SH3CIN85
wild-type
targeted
floxed
deleted
N N N
1kb Probe
N loxP N
neor
N
N N
N N
5 kb
3.3 kb
6.8 kb
4.8 kb
2R
C9 C10
2R
C10
CIN85- CIN85+
5 kb
3.3kb
X chromoseome
CIN85 flox
WT CIN85-
CD4
TCR b
1 10 100 1 0 00 1 0 00 0
P e rCP - C y 5 - 5- A
1
10
100
1 0 00
1 0 00 0
A
P
C-A
4,3 8 7 7,8
1 4,63,2 6
1 10 100 1 0 00 1 0 00 0
P e rCP - C y 5 - 5- A
1
10
100
1 0 00
1 0 00 0
A
P
C-A
3,1 7 8 1,6
1 2,23,0 4
1 10 100 1 0 00 1 0 00 0
F IT C- A
1
10
100
1 0 00
1 0 00 0
P
E
-A
0,2 3 0,0 1 3
2 5,57 4,2
1 10 100 1 0 00 1 0 00 0
F IT C- A
1
10
100
1 0 00
1 0 00 0
P
E
-A
0,1 9 0,0 1 1
2 0,47 9,4
1 10 100 1 0 00 1 0 00 0
F IT C- A, c f s e F I T C- A
0
20
40
60
80
100
%
o
f
M
a
x
1 10 100 1 0 00 1 0 00 0
F IT C- A, c f s e F I T C- A
0
20
40
60
80
100
%
o
f
M
a
x
1 10 100 1 0 00 1 0 00 0
F IT C- A, c f s e F I T C- A
0
20
40
60
80
100
%
o
f
M
a
x
1 10 100 1 0 00 1 0 00 0
F IT C- A, c f s e F I T C- A
0
20
40
60
80
100
%
o
f
M
a
x
WT
CIN85-
4.5 day CFSE
CD4 CD8
CIN85是 T 细胞受体信号
通路的调控蛋白。用荧光
染料 CFSE标记从 CIN85
基因剔除小鼠和野生型小
鼠的脾脏中分离 T淋巴细
胞，并用 5 mg/ml CD3进
行刺激,4.5天后，用流
式细胞仪分析 T细胞受体
介导的细胞增殖。细胞内
CFSE的荧光强度随细胞
分裂而减弱，结果显示
CIN85基因剔除的 CD4和
CD8 T细胞分裂得更快。
non-sti
sti
谢谢！
