遗传工程小鼠的制备繁育及科学应用

6
Knock-in
* loxP
loxP
12
345
NeoR
pA
6
KO first, Conditional Ready Allele
FRT
loxP
12
SA IRES
lacZ
pA
NeoR
FRT loxP
loxP
pA
345
6
Story 2 蛋白磷酸化调控
PI3K-PKB/Akt Pathway in Cardiac system
The “conventional” transgenesis is using short constructs with well-defined promoter regions.
Evans Cappechi
2007年诺贝尔生理医学奖
第一个基因敲除小鼠（1987-89）
2006年 EUCOMM: 1.4亿欧元 NorCOMM: 1 亿加元 KOMP: 1.8亿美元wk.baidu.comChCOMM: 1500万RMB
“I don’t think there’s any example of any field moving this fast”
----Blake Wiedenheft Montana State University
How CRISPR-Cas9 works?
CREDIT: K. SUTLIFF/SCIENCE
Transgene integration site
LC
RC1
RC2 RC3
Sha, H., et al (,2007) Physiol Genomics. 31(2): p. 252-63
第一个转基因小鼠（1982）
by Richard Palmiter and Ralph Brinster
Procedure of conventional TG
Challenges in Chemotherapy and the Drug development
Low response rate of SOC to different persons
Success rate by stages of clinical testing for oncology compounds.
nervous system
Lung Development
Ureter Development
The CRISPR Craze
Science 23 August 2013 vol. 341
NEWS FOCUS
The CRISPR Craze
A bacterial immune system yields a potentially revolutionary genome-editing technique
Progress of NBRI’s CRISPR/Cas9 Service
➢The first cKO mouse in china(2005) ➢The first single and multiple genes Cas9 KO mouse in china(2013) ➢The first Cas9 cKO rat in china(2013) ➢The first KO monkey in the world (2015) ➢The first KO dog in the world (2015)
基因组改造技术是建立疾病模型的核心
Smithies
ES target strategy
Wildtype Allele
12
345
6
Knockout Allele
loxP
12
NeoR
loxP
pA
6
Conditional KO Allele (flox)
loxP
FRT
12
345
NeoR
FRT loxP
pA
PKB alpha PKB beta PKB gama
PP2A
PDK1 (cKO, TM)
出生后心脏发育不良和心衰
心脏ASD(房室隔缺陷)
Yin et al, Genes Dev, 2012; Di et al, Biochem. J., 2012; Fang et al, Mol Cell Biol, 2010; Zhou et al, Am J Path, 2010;
遗传工程小鼠的制备繁育及应用
Story 1 Timo “transgenic” mice
(transgenic insertional mutants with obesity)
Timo “transgenic” mice
Mouse Ch2
~7kb
Regulatory locus BDNF/Bdnf Transgene insertion
A better pre-clinical evaluating system is needed
Zhang et al, J. Pathol., 2012 Lu et al, Plos ONE, 2011 Chang et al, Dev Biol, 2010; Chang et al, Front Biosci, 2010
故事三 中国第一个条件性敲除模型（2005） Fstl1, Follistatin-like 1
Animals distribution
Training
➢Customized TG/KO Mouse
➢ Mouse phenotype
Breeding services
Import / export
Rederivation & cryopreservation
Veterinary services
12
345
6
Conditional KO Allele (flox)
loxP
loxP
12
345
6
Knock-in
*
12
345
6
Nanjing Biomedical Research Institute
of Nanjing University (NBRI)
Resource +
Research
One-Stop Services
Cas9技术流程
KO ：1 week cKO/KI：1-2 months
F0 generation ： 1 month F1 generation ： 3 months
无ES细胞限制，可选用小鼠品系多 GLT率高，快速获得杂合子（6 months）
Cas9 cKO/KI strategy
Wildtype Allele