基因打靶定点突变秦川牛MSTN基因.pdf

生 物 工 程 学 报 Chin J Biotech 2010, March 25; 26(3): 410–416 journals.im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061 cjb@im.ac.cn ©2010 CJB, All rights reserved. 生物技术与方法 MSTN 刘永刚，华松，兰杰，宋永利，何玉龙，权富生，张涌 712100 ，肌肉生长抑制素 基因属于 超家族，对骨骼肌的生长发育具有负调控作用。该基因 : Myostatin (MSTN ) TGF-β 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 、测序及 鉴定，结果显示，第 号细胞克隆为发生了正 PCR Southern blotting 58 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。 : 肌肉生长抑制素，基因打靶，定点突变，成纤维细胞，秦川牛 Site-directed mutagenesis of MSTN gene by gene targeting in qinchuan cattle Yonggang Liu, Song Hua, Jie Lan, Yongli Song, Yulong He, Fusheng Quan, and Yong Zhang Key Laboratory of Animal Reproductive Endocrinology Embryo Engineering, Ministry of Agriculture, College of Veterinary Medicine, Northwest A F University , Yangling 712100, China 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