CRISPR/Cas9介导CPED1基因敲除载体活性的验证  

Verification of CPED1 Gene Knockout Vector Activity by CRISPR/Cas9

作　　者：王飞[1] 何娜娜[1] 王颖洁[1] 纪艳芹 张亚妮[1] 李碧春[1] 

机构地区：[1]扬州大学动物科学与技术学院,江苏省动物繁育与分子设计重点实验室,江苏扬州225009

出　　处：《中国家禽》2017年第7期11-14,共4页China Poultry

基　　金：国家自然科学基金项目(31472087)

摘　　要：试验旨在利用CRISPR/Cas9技术分别在鸡成纤维细胞DF-1和鸡胚胎干细胞(Em-bryomic stem cell,ESCs)上介导CPED1基因敲除,以期为后续研究CPED1基因功能提供技术依据。克隆CPED1基因全长;构建cas9/g RNA载体并转染DF-1和ESCs,经流式分选阳性细胞,采用T7E1酶切法选择活性最佳的敲除载体并分析其敲除效率;同时,对DF-1中的脱靶效率进行检测。结果成功克隆鸡CPED1基因,并构建3个cas9/g RNA载体;T7E1酶切结果表明cas9/g RNA1载体在DF-1阳性细胞中的敲除活性(37%)最佳,该载体也可定点敲除ESCs中的CPED1基因,敲除活性为25%,且在DF-1细胞中未发生脱靶。表明CRISPR/Cas9可有效介导鸡CPED1基因在DF-1和ESCs上敲除。This was study aimed to investigate the effect of CPED1 gene knockout on chicken fibroblasts （DF-1） and chicken embryonic stem cells （ESCs）with gene edition technique mediated by CRISPR/Cas9,respectively. In order to provide technical basis for further investigate the function of CPED1 gene,the full length of CPED1 gene and construct eas9/gRNA vectors were cloned. Then both DF-1 and ESCs were transfected with gRNA vector,and screened by flow cytometry. The optimal knockout vector was selected by T7E1 digestion and the knockout efficiency was analyzed. At the same time,the efficiency of the off-targe in DF-1 cells was detected. The knockout target was used to detect the off-target efficiency in DF-1 cells. The results showed that CPEDI gene was successfully cloned and the cas9/gRNA vectors were constructed. The results of T7E1 digestion showed that the knockout activity of eas9/gRNA1 vector in DF-1 positive cells was the best （37%）. The knockout activity of CPED1 gene in the ESCs was determined to be 25%,and the target didn＇t occur in DF-1 cells. It＇s suggested that CRISPR/Cas9 could effectively induce chicken CPED1 gene knockout on DF-1 and ESCs.

关 键 词：CRISPR/Cas9技术 CPED1基因 鸡 敲除载体 

分 类 号：S831.2[农业科学—畜牧学]