利用TALEN技术在牛胎儿成纤维细胞中敲除Myostatin基因

doi:10.3724/SP.J.1005.2014.0685

遗传 ›› 2014, Vol. 36 ›› Issue (7): 685-690.doi: 10.3724/SP.J.1005.2014.0685

利用TALEN技术在牛胎儿成纤维细胞中敲除Myostatin基因

杨翠翠, 佟慧丽, 马兴红, 杜巍, 刘丹, 杨宇, 严云勤

东北农业大学生命科学学院, 哈尔滨 150030

收稿日期:2013-12-16 出版日期:2014-07-20 发布日期:2014-06-23
通讯作者: 严云勤, 教授, 博士生导师, 研究方向：转基因动物。E-mail: yanyunqin@sohu.com
作者简介:杨翠翠, 硕士, 专业方向：分子克隆与基因敲除。E-mail: 806344748@qq.com
基金资助:
国家转基因专项(编号：2011ZX08007-002)资助

Myostatin knockout in bovine fetal fibroblasts by using TALEN

Cuicui Yang, Huili Tong, Xinghong Ma, Wei Du, Dan Liu, Yu Yang, Yunqin Yan

College of Life Science, Northeast Agricultural University, Harbin 150030, China

Received:2013-12-16 Online:2014-07-20 Published:2014-06-23

摘要/Abstract

摘要： 肌肉生长抑制素(Myostatin, MSTN)基因能够负向调节骨骼肌的生长和发育, 牛MSTN基因突变会出现“双肌”特征。文章利用转录激活因子样效应物核酸酶(TALENs)靶向敲除牛胎儿成纤维细胞的MSTN基因, 获得敲除MSTN基因的细胞系, 为制备MSTN基因敲除牛提供材料。构建一对MSTN基因的TALENs真核表达载体, 分别采用PEI转染试剂和电穿孔法进行牛胎儿成纤维细胞的转染, 测序结果表明TALEN技术可用于敲除牛MSTN基因, 利用T7核酸内切酶1(T7E1)检测其突变效率, 结果显示电穿孔转染的敲除效率为20.4%。通过有限稀释法, 共获得10个MSTN基因敲除的细胞克隆(包括MSTN^-/-和MSTN^+/-), 其靶位点敲除的碱基数分别是1~20不等, 部分会出现移码突变。出现移码突变的细胞系可用于MSTN基因敲除的转基因肉牛的制备。

关键词: TALENs, 牛, 敲除, 转染

Abstract: Myostatin (MSTN) can negatively regulate the growth and development of skeletal muscle, and mutations of bovine MSTN gene can cause a “double-muscle” feature. To knock out MSTN gene in bovine fetal fibroblast by transcription activator-like effector nucleases (TALENs) and obtain MSTN knockout cell lines, we constructed one pair of MSTN-TALEN vector and transfected into bovine fetal fibroblast cells by PEI and electroporation. Sequencing results demonstrated that TALEN was available for MSTN knockout. T7 endonuclease 1 (T7E1) was used for the detection of mutation efficiency. The results indicated that knockout efficiency of electroporation transfection was 20.4%, and 10 MSTN^+/- and MSTN^-/- cell colonies were obtained via limiting dilution method. The deletion number of nucleotides ranged from 1 to 20, and some of them were frameshift mutation, which could provide the possibility in production of MSTN knockout cattle in the future.

Key words: TALENs, bovine, knockout, transfection

杨翠翠, 佟慧丽, 马兴红, 杜巍, 刘丹, 杨宇, 严云勤. 利用TALEN技术在牛胎儿成纤维细胞中敲除Myostatin基因[J]. 遗传, 2014, 36(7): 685-690.

Cuicui Yang, Huili Tong, Xinghong Ma, Wei Du, Dan Liu, Yu Yang, Yunqin Yan. Myostatin knockout in bovine fetal fibroblasts by using TALEN[J]. HEREDITAS(Beijing), 2014, 36(7): 685-690.

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利用TALEN技术在牛胎儿成纤维细胞中敲除Myostatin基因

Myostatin knockout in bovine fetal fibroblasts by using TALEN

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