利用CRISPR/Cas9系统敲除绵羊皮肤成纤维细胞Wnt2基因的研究  

Knockout of Wnt2 in the primary cell of sheep skin fibroblast via the CRISPR/Cas9 system

作　　者：冯新宇 袁超[1] 郭婷婷[1] 刘建斌[1] 郭健[1] 牛春娥[1] 孙晓萍[1] 冯瑞林[1] 岳耀敬[1] 杨博辉[1] 

机构地区：[1]中国农业科学院羊育种工程技术研究中心/中国农业科学院兰州畜牧与兽药研究所

出　　处：《畜牧与兽医》2018年第2期52-55,共4页Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学青年基金(31402057);中国农业科学院细毛羊资源与育种创新工程(CAAS-ASTIP-2015-LIHPS-03);国家绒毛用羊产业技术体系分子育种岗位(No.nycytx-40)

摘　　要：为了研究无翅基因相关整合位点2（Wnt2）在绵羊毛囊发育中的作用,通过CRISPR/Cas9系统介导的基因编辑技术在绵羊原代皮肤成纤维细胞中敲除Wnt2基因,经T7E1酶切试验进行敲除结果验证,实时荧光定量PCR分析Wnt2、E-钙黏蛋白（CDH1）、半光天冬氨酸酶3（Caspase3）和成纤维细胞生长因子5（FGF5）在敲除后的表达量变化。结果显示：绵羊原代皮肤成纤维细胞中Wnt2基因敲除成功,编辑效率约14.35%;Wnt2相对表达量极显著下调（P〈0.01）,CDH1相对表达量极显著上调（P〈0.01）,Caspase3及FGF5表达上调显著（P〈0.05）。结果表明：Wnt2基因对毛囊的生长发育具有促进作用。The CRISPR/Cas9 system was used in the present experiment to knockout Wnt2 in the primary cell of sheep skin fibroblast.T7 E1 enzyme digestion was employed to verify the knockout result. And RT-PCR was performed to analyze differences in the expression of Wnt2,CDH1,caspase3 and FGF5 after knockout. The results show that the Wnt2 knockout was successful with the editing efficiency being14. 35%. The expression of Wnt2 was down-regulated very significantly（P〈0. 01）,and the expression of CDH1 was up-regulated very significantly（P〈0. 01）. The expressions of FGF5 and caspase3 were significantly up-regulated（P〈0. 05）. To conclude,Wnt2 promotes the growth and development of hair follicles.

关 键 词：无翅基因相关整合位点2 毛囊 CRISPR/Cas9 RT-PCR 

分 类 号：S826[农业科学—畜牧学]