CRISPR/Cas9介导的绵羊胎儿成纤维细胞肌肉生长抑制素基因敲除研究  

Knockout of Myostatin Gene by CRISPR/Cas9 System in Ovine Fetal Fibroblasts

作　　者：李聪[1] 曹文广[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《中国畜牧兽医》2015年第11期2813-2821,共9页China Animal Husbandry & Veterinary Medicine

基　　金：国家转基因重大专项(2013ZX08008-003;2014ZX08008-003)

摘　　要：肌肉生长抑制素(myostatin,MSTN)基因突变可引起动物出现"双肌"性状,提高产肉性能。利用CRISPR/Cas9技术制备MSTN基因敲除的绵羊胎儿成纤维细胞,为制备MSTN基因敲除羊提供材料。设计构建4个靶向MSTN基因的CRISPR/Cas9载体,脂质体转染细胞后,通过SURVEYOR分析和测序等方法对敲除效率进行检测,采用极限稀释法挑选稳定敲除的细胞系。试验成功构建了4个靶向MSTN基因的CRISPR/Cas9载体,细胞转染后,测序结果显示pX330-target 1和pX330-target 4载体作用的靶位点处出现突变,SURVEYOR分析检测其在靶位点产生切割的效率分别为24.20%和10.18%。通过极限稀释法,获得12个MSTN基因突变的细胞克隆,其中1个为纯合突变。序列比对发现靶位点处有小片段碱基插入或缺失突变,部分会出现移码突变。成功利用CRISPR/Cas9系统实现了绵羊MSTN基因敲除,证明该系统可有效应用于绵羊基因编辑,产生的突变细胞系为制备MSTN基因敲除羊提供了材料。Mutation in myostatin（MSTN）gene resulted in double muscle effect,generating more mutton.To knock out MSTN gene in sheep fetal fibroblast by CRISPR/Cas9 system and obtain MSTN gene knockout cell lines,four plasmids were designed and constructed to target MSTN gene,and confirmed correctly by sequencing.The correct plasmids were delivered into the fetal fibroblast cells.The targeting efficiency was detected using SURVEYOR assay Kit.The stable transfected cell colonies were obtained via limiting dilution procedure.The sequence results demonstrated that the pX330-target 1 and pX330-target 4 plasmids could successfully knockout MSTN gene,and the targeting efficiency were 24.20%and 10.18%,respectively.Twelve MSTN gene knockout cell colonies were obtained via limiting dilution,and one of them was homozygous mutation.Several indel mutations were discovered at specific site,and some of them were frameshift mutation.Therefore,we concluded that the CRISPR/Cas9 system could apply to the gene editing of sheep efficiently,and the gene knockout cell clones had potential application in generating MSTN gene knockout sheep.

关 键 词：CRISPR/Cas9系统 肌肉生长抑制素 基因敲除 绵羊 

分 类 号：Q78[生物学—分子生物学]