基因打靶定点突变秦川牛MSTN基因

Site-directed mutagenesis of MSTN gene by gene targeting in qinchuan cattle

摘要:

Myostatin (MSTN，肌肉生长抑制素) 基因属于TGF-β超家族，对骨骼肌的生长发育具有负调控作用。该基因的功能缺失，能够引起肉用动物的“双肌”表型，从而提高产肉率。基因打靶技术是制作转基因动物的常用方法。构建了两个置换型打靶载体pA2T-Mstn4.0和pA2T-Mstn3.2，通过同源重组将G938A突变点引入秦川牛MSTN基因第三外显子。电穿孔方法转染秦川牛胎儿成纤维细胞，经过600 μg/mL G418和50 nmol/L GCV的药物正负筛选，共得到170个药物抗性细胞克隆。对细胞克隆进行PCR、测序及Southern blotting 鉴定，结果显示，第58号细胞克隆为发生了正确同源重组的中靶细胞。牛胎儿成纤维细胞中的MSTN基因的一条等位基因被成功改造。

Abstract:

Myostatin, a member of the transforming growth factor β (TGF-β) family, is a negative regulator for muscle growth. Loss of the function of this gene is associated with the phenotype described as “double muscling”, an extreme form of muscle development characterized by a large increase in muscle mass. Two replacement vectors, pA2T-Mstn4.0 and pA2T-Mstn3.2, were constructed, linearized, and transfected into the bovine fetal fibroblasts through electroporation. 170 drug-resistant cell colonies were obtained in cell culture medium containing 600 μg/mL G418 and 50 nmol/L GCV. Targeted homologous integration occurred in colony No. 58 as identified by PCR, and the targeted colony was further confirmed by sequencing and Southern blotting. This suggested that one allele of myostatin was successfully mutagenized in bovine fetal fibroblasts.