利用CRISPR/Cas9技术制备敲除Myostatin基因牛胎儿成纤维细胞株  

Knockout of the myostatin gene to prepare bovine fetal fibroblast line using CRISPR / Cas9 technology

作　　者：杨宇[1] 张伟伟[1] 胡倩[2] 佟慧丽[1] 严云勤[1] 

机构地区：[1]东北农业大学生命科学学院,哈尔滨150030 [2]东北农业大学动物医学学院,哈尔滨150030

出　　处：《黑龙江畜牧兽医》2015年第9期50-53,共4页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家转基因重大专项"高产优质转基因肉牛新品种培育"(2014ZX08007-002)

摘　　要：CRISPR/Cas9系统作为第3代人工核酸内切酶,是一种具有广阔应用前景的基因定点修饰工具。肌肉生长抑制素(Myostatin,MSTN)基因能够负向调节肌肉的生长,影响动物的产肉量。为了获得MSTN基因敲除的细胞株,给转基因动物的制备提供材料,试验利用CRISPR/Cas9系统的不同类型的表达载体对牛MSTN基因外显子序列随机设计靶位点,利用T7核酸内切酶I(T7EI)从中挑选出敲除效率最高的靶位点的表达载体,在无筛选标记条件下对牛胎儿成纤维细胞进行基因敲除。结果表明:经过长时间培养获得了基因敲除的细胞株。说明CRISPR/Cas9技术用于基因敲除方面的研究应用前景广阔。CRISPR/Cas9 system as the third generation of artificial endonuclease, is a promising tool for gene site -specific modification. Myostatin （MSTN） gene can negatively regulate muscle growth to affects animal＇ s meat production, in order to obtain the MSTN gene knockout cell line and provide materials to the preparation of transgenic animals, different iypes of expression vectors of CRISPR/Cas9 system were utilized to randomly design of the target sites for exon sequences of MSTN gene. T7 endonuclease I （T7EI） was utilized to select the expression vector with the highest removal efficiency of the target sites. The gene knockout was carried out in the bovine fetal fibroblast cells under the condition of without screening markers, The results showed that the gene knockout cell line was obtained through the long-time culture. The result indicates that CRISPR/Cas9 technology has wide application prospect in geue knockout.

关 键 词：CRISPR/Cas9 肌肉生长抑制素(Myostatin MSTN) 转染 敲除 突变效率 细胞株 

分 类 号：S823[农业科学—畜牧学] S813.1[农业科学—畜牧兽医]