《中国畜牧兽医》 ›› 2018, Vol. 45 ›› Issue (4): 841-849.doi: 10.16431/j.cnki.1671-7236.2018.04.001

• 生物技术 •    下一篇

CRISPR-Cas9介导的蒙古牛MSTN基因敲除细胞系的建立

郭梓茹, 张立, 马云龙, 苏小虎, 周欢敏, 张焱如, 刘晓   

  1. 内蒙古农业大学生命科学学院, 呼和浩特 010018
  • 收稿日期:2017-09-08 出版日期:2018-04-20 发布日期:2018-04-25
  • 通讯作者: 张立(1961-),男,内蒙古呼和浩特人,博士,研究方向:动物细胞调控及胚胎工程,E-mail:zhanglinmg@aliyun.com E-mail:zhanglinmg@aliyun.com
  • 作者简介:郭梓茹(1990-),女,山西忻州人,硕士,研究方向:动物细胞调控及胚胎工程,E-mail:534518154@qq.com
  • 基金资助:

    转基因体细胞克隆牛妊娠维持相关microRNA的筛选及鉴定(31460599)

Establishment of MSTN Gene Knockout in Mongolia Bovine Cell Lines Mediated by CRISPR-Cas9 System

GUO Ziru, ZHANG Li, MA Yunlong, SU Xiaohu, ZHOU Huanmin, ZHANG Yanru, LIU Xiao   

  1. College of Life Science, Inner Mongolia Agricultural University, Hohhot 010018, China
  • Received:2017-09-08 Online:2018-04-20 Published:2018-04-25

摘要:

试验旨在利用CRISPR-Cas9技术对蒙古牛胎儿成纤维细胞肌肉生长抑制素(myostatin,MSTN)基因进行编辑,构建无标记的MSTN基因敲除的蒙古牛细胞系,为培育肌肉发达的蒙古牛提供试验材料。通过组织块贴壁法对妊娠45 d的蒙古牛胎儿建立皮肤成纤维细胞系,同时在第2外显子区域选定1个gRNA靶位点,使用在线软件设计sgRNA序列,并选取评分较高的3个sgRNA,采用试剂盒法构建无标记的Cas9/gRNA载体,随后用T7E1酶酶切法对其进行活性验证,将有活性的载体用电转法转染蒙古牛胎儿成纤维细胞,通过无限稀释法和口吸管法将转染后的单细胞接种于96孔板扩繁后提取DNA,对其进行PCR扩增及测序验证来获取阳性细胞。本试验构建了3个无标记的CRISPR-Cas9载体,经验证1个有活性,对电转染后的单细胞进行筛选共获得96个单细胞株,测序分析后有1个单细胞株发生单碱基突变,使得MSTN基因不能编码正常的蛋白。试验成功建立了1个MSTN基因失活的细胞株,可作为后续体细胞核移植的供体细胞,用于生产无标记的敲除MSTN基因的蒙古牛,对培养产肉率高并具有生物安全性的蒙古牛具有重要的意义。

关键词: CRISPR-Cas9系统; MSTN基因; 蒙古牛; 无标记细胞系

Abstract:

This study was aimed to edit myostatin (MSTN) gene of Mongolian bovine fetal fibroblasts using CRISPR-Cas9 system,build an unmarked Mongolian bovine cell line which its MSTN gene had been knocked out,and provide experimental material for cultivating muscular Mongolian bovine.Skin fibroblast cell lines were established on 45 day of gestation in Mongolia bovine fetus by tissue adherent method,at the same time,a gRNA target site in exon 2 region was selected,and three high scores sgRNAs sequences which designed in online software were chosen,unmarked Cas9/gRNA vector was constructed by kit method,its activities was tested by T7E1 digestion,the active vector was transfected into Mongolian bovine fetal fibroblasts by electric transfer,and seed transfected single cells in 96-well plates by infinite dilution method and pipette method,which DNA was extracted,and obtained positive cells by PCR amplification and sequencing verification in the end.The results showed that we constructed three unmarked CRISPR-Cas9 vectors and found that only one vector was active.A total of 96 single cell strain were obtained by screening single cells which after electrical transfection,and only one single cell strain was found that occur single-base mutation after sequencing analysis,which made MSTN gene could not encode normal protein.This study established successfully a MSTN gene inactivated cell line,which could be used as a donor cell for subsequent somatic cell nuclear transfer for the production of unmarked Mongolian bovine with MSTN gene knockouted.It was of great significance to cultivate Mongolian bovine with high meat production rate and biosecurity.

Key words: CRISPR-Cas9 system; MSTN gene; Mongolia bovine; non-lable cell line

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