中山大学学报自然科学版

• 研究论文 • 上一篇    下一篇

利用CRISPR/Cas9技术敲除小鼠ES细胞H2-K1基因

陈瑞俊1, 李蔚然1, 黄乙涓1 , 刘建中1, 李亮平1,2   

  1. 1 中山大学中山医学院生物学教研室,广东 广州 510080;
    2. 暨南大学附属第一医院临床医学研究院,广东 广州 510632
  • 收稿日期:2016-09-05 出版日期:2017-03-25 发布日期:2017-03-25
  • 通讯作者: 李亮平(1961年生),男;研究方向:肿瘤免疫学;E-mail:lilping2@mail.sysu.edu.cn,liangping_li@jnu.edu.cn

The H2-K1 gene knockout of mouse ES cells through CRISPR/Cas9 technology

CHEN Ruijun1,LI Weiran1,HUANG Yijun1,LIU Jianzhong1,LI Liangping1,2   

  1. 1.Department of Biology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China; 
    2.Central Laboratory, The First Affiliated Hospital, Jinan University, Guangzhou 510632, China
  • Received:2016-09-05 Online:2017-03-25 Published:2017-03-25

摘要:

利用CRISPR/Cas9和ES细胞技术,在小鼠ES细胞株上敲除小鼠H2-K1基因,为研发MHCⅠ类基因人源化小鼠打下基础。设计了两个单向导RNA(single guide RNA,sgRNA),分别靶向H2-K1基因的外显子2和外显子3。以pX330质粒为骨架构建表达sgRNA的打靶载体。用电穿孔转染法将构建好的质粒以及pSUPERpuro共同导入小鼠ES细胞中,实现基因敲除。在嘌呤霉素筛选后,利用PCR初步检测靶基因的敲除情况,再通过测序以及流式细胞分析确定敲除H2-K1基因的小鼠ES细胞。结果显示:利用CRISPR/Cas9技术,小鼠ES细胞的H2-K1基因被成功敲除,通过PCR检测到4个克隆为H2-K1单等位基因敲除(19.0%),2个克隆为H2-K1双等位基因敲除(9.5%)。经过测序以及流式细胞分析,2株小鼠ES细胞被确认为H2-K1双等位基因敲除。本研究利用CRISPR/Cas9技术得到H2-K1基因敲除的小鼠ES细胞株, 为MHCⅠ类基因的敲除和置换提供了参考。

关键词: H2-K1 基因, CRISPR/Cas9技术, mES细胞, 基因敲除

Abstract:

The CRISPR/Cas9 technology was used to achieve the H2-K1 gene knockout of mouse ES cells, which will be useful for generation of MHC class Ⅰ gene humanized mice. In this study, two sgRNAs were designed, which are targeting to the Exon2 and Exon3 of H2-K1, respectively. The plasmids expressing the sgRNAs were constructed using pX330 as the matrix plasmid. In order to knockout H2-K1, the constructed plasmids and pSUPERpuro were cotransfected into the mES cells through electroporation. After screening by puromycin, the result of gene targeting was determined by PCR,and H2-K1 knockout mouse ES cells were further confirmed through sequencing and flow cytometry. We found that H2-K1 in the mouse ES cell was knocked out using CRISPR/Cas9 technology. Through PCR, 4 clones were determined as one allele knockout(19.0%),2 clones were determined as two allele knockout(9.5%). 2 clones were further confirmed as two allele knockout clones by sequencing and flow cytometry. The generated H2-K1 knockout mouse ES cells would provide a reference for the knockout and replacement of MHC class Ⅰ gene.

Key words: H2-K1 gene, CRISPR/Cas9 technology, mES cell, gene knockout

中图分类号: