R26-CAG-2xKRAB-dCas9-IRES-EGFP

品系全名

C57BL/6Smoc-Gt(ROSA)26Sorem1(CAG-Loxp-2xKRAB-dCas9-IRES-EGFP)Smoc

目录号

NM-KI-18007

品系状态

活体

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基因信息

基因名
Gt(ROSA)26Sor

基因曾用名

R26，ROSA26，AV258896，Gtrgeo26，Gtrosa26，Thumpd3as1

NCBI ID

14910

MGI ID

104735

Ensembl ID

ENSMUSG00000086429

Pubmed

Gt(ROSA)26Sor

品系描述

将CAG-LSL-2xKRAB-dCas9-IRES-EGFP-WPRE-pA插入到小鼠Rosa26基因中，建立CRISPRi基因表达调控工具小鼠。该小鼠已去掉stop。 CRISPRi是基于一个没有核酸酶活性的dCas9蛋白，当dCas9被引导到某个基因的转录起始位点TSS（transcription start site）时，dCas9能够物理性阻碍RNA聚合酶的通过，导致基因沉默。为了进一步提高转录抑制的效率，dCas9可以融合一些基因抑制结构域，如KRAB（krüppel-associated box）结构域，强化抑制效果。
应用领域：CRISPRi,基因调控工具

验证数据

通过Westernblot验证2xKRAB-dCas9表达


Rosa26- CAG-LSL-2xKRAB-dCas9 mice were crossed with Dppa3-Cre mice to delete STOP cassette.


Anti-Cas9 (1).jpg

图1 该小鼠含有loxp-STOP-loxp结构，是条件性过表达2XKRAB-dCas9，需要和Cre交配才能表达2XKRAB-dCas9 。该小鼠和广泛表达的Cre(Dppa3-Cre)交配后检测Cas9表达，westernblot检测发现Cas9在肝脏、脾、肺、肾、胸腺、皮质和小脑中都高表达。

通过荧光测定验证EGFP表达

CRISPRi—inhibitor： dCas9-KRAB_06.png

图2 该小鼠和广泛表达的Cre（Dppa3-Cre）交配后检测EGFP表达，检测发现大脑、肺、肾、胸腺、肝脏中EGFP表达较强。

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