利用CRISPR/Cas9敲除大鼠胰岛素受体底物1(Irs1)基因  

Generating insulin receptor substrate 1( Irs1) knockout rat using CRISPR /Cas9

作　　者：马元武[1] 马婧[1] 路迎冬[1] 陈炜[1] 张旭[1] 于磊[1] 张连峰[1] 

机构地区：[1]中国医学科学院,北京协和医学院,医学实验动物研究所,卫生部人类疾病比较医学重点实验室,北京100021

出　　处：《中国比较医学杂志》2014年第3期55-60,共6页Chinese Journal of Comparative Medicine

基　　金：国家科技支撑计划课题(2012BA139B02)

摘　　要：目的为研究胰岛素受体底物1(Irs1)基因与代谢病之间的关系,我们利用CRISPR/Cas9系统敲除大鼠Irs1基因,为研究代谢病提供基因敲除大鼠。方法针对Irs1第一外显子,设计CRISPR/Cas9作用靶点,构建sgRNA表达质粒。利用T7 RNA聚合酶体外转录sgRNA和Cas9。将Cas9 mRNA和sgRNA混合物注射入SD大鼠的受精卵中,实现靶基因敲除。用T7EN1实验初步检测靶基因的修饰情况,再经过测序分析确定突变。结果获得了5个在Irs1基因突变的首建鼠,突变效率为83%。结论得到了稳定遗传的Irs1基因敲除大鼠。Objective To study the relationship of insulin receptor substrate-1 （Irs1） and metabolic disease, we generated Irs1 gene knockout rat by CRISPR/Cas9 system.Methods Two sgRNA targeting sites were designed for Irs1 targeting.The Cas9 and sgRNAs were transcribed by T7 RNA polymerase in vitro.Cas9 mRNA and sgRNA mixtures were pooled and microinjected into one-cell fertilized eggs of SD rats to generate rats with targeted mutation .Results Five rats with the mutations were detected with the efficiency of 83%.Conclusion The Irs1 gene knockout rats generated in this study can be transmitted by germline .

关 键 词：Irs1 基因敲除 大鼠 CRISPR Cas9 

分 类 号：R332[医药卫生—人体生理学]