研究报告

利用TALEN技术在牛胎儿成纤维细胞中敲除Myostatin基因

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  • 东北农业大学生命科学学院, 哈尔滨 150030
杨翠翠, 硕士, 专业方向:分子克隆与基因敲除。E-mail: 806344748@qq.com

收稿日期: 2013-12-16

  网络出版日期: 2014-06-23

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

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

Received date: 2013-12-16

  Online published: 2014-06-23

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; ; 敲除; 转染

本文引用格式

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 . DOI: 10.3724/SP.J.1005.2014.0685

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.

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