生物技术通报 ›› 2019, Vol. 35 ›› Issue (11): 240-250.doi: 10.13560/j.cnki.biotech.bull.1985.2019-0426

• 技术与方法 • 上一篇    下一篇

应用CRISPR技术敲除MDA-MB-231细胞系的NODAL基因的研究

李念峰, 方天星, 倪玉芳, 曾凡才   

  1. 西南医科大学基础医学院生物化学与分子生物学实验室,泸州 646000
  • 收稿日期:2019-05-17 出版日期:2019-11-26 发布日期:2019-11-19
  • 作者简介:李念峰,男,硕士研究生,研究方向:肿瘤分子生物学;E-mail:913375573@qq.com
  • 基金资助:
    四川省科技厅-泸州市-泸州医学院联合基金(LY-100),四川省科技厅基金(2013SZZ001)

Knockout of NODAL Gene in MDA-MB-231 Cell Line by CRISPR/Cas9

LI Nian-feng, FANG Tian-xing, NI Yu-fang, ZENG Fan-cai   

  1. Laboratory of Biochemistry and Molecular Biology,School of Basic Medical Sciences,Southwest Medical University,Luzhou 646000
  • Received:2019-05-17 Published:2019-11-26 Online:2019-11-19

摘要: NODAL(Nodal growth differentiation factor)是一种细胞因子,为研究其功能,需建立敲除NODAL基因的细胞系。构建了两种作用于不同靶位点的CRISPR/Cas9表达载体和一种打靶载体,并通过电转法导入三阴性乳腺癌细胞系MDA-MB-231,经嘌呤霉素抗性筛选和挑单细胞克隆,再应用PCR和测序技术鉴定基因型,以及Western Blot检测蛋白表达。将等位基因同源重组鉴定为阳性的8个单细胞克隆再经非同源重组和大片段缺失基因型鉴定,仅一个单细胞克隆的靶位点发生移码突变,4个单细胞克隆的靶位点有大片段缺失发生,4个单细胞克隆的靶位点检测到野生型等位基因。蛋白表达检测结果显示,仅有2个单细胞克隆未检测到NODAL蛋白表达,3个单细胞克隆的NODAL蛋白表达降低,另外3个单细胞克隆的NODAL蛋白表达无明显变化。最终获得了基因型和蛋白表达鉴定为敲除NODAL基因的单细胞克隆,为进一步研究NODAL在乳腺癌细胞中的分子机制奠定了基础。

关键词: CRISPR/Cas9, 乳腺癌, 基因敲除, NODAL基因

Abstract: In order to study the function of growth differentiation factor NODAL(nodal growth differentiation factor),the cell line with NODAL gene knockout needs to be established. Two different CRISPR/Cas9 expression vectors targeting different sites and one targeting vector for homologous recombination were constructed and then transfected into triple negative breast cancer cell line MDA-MB-231 by electrotransfection. The genotypes after purinomycin resistance screening and single-cell clones selection were identified by PCR and sequencing,and protein expression was detected by Western blot. Eight single-cell clones identified as positive by allelic homologous recombination assay were re-identified by non-homologous recombination and large fragment deletion analyses. Only one single-cell clone had a frame-shift mutation in the target site. A large fragment deletion occurred at the target sites of the 4 single-cell clones,and the wild-type alleles were detected at the target sites of the 4 single-cell clones. Western blot showed that NODAL expressions in only 2 single-cell clones were not detected,and NODAL expressions were decreased in 3 single-cell clones,and no significant changes in NODAL expressions were found in other 3 single-cell clones. Thus,the single-cell clones with NODAL gene knockout are obtained based on genotype identification and Western blot,which lays a foundation for further research on the molecular mechanism of NODAL in breast cancer cells.

Key words: CRISPR/Cas9, breast cancer, gene knockout, NODAL gene