应用CRISPR技术敲除MDA-MB-231细胞系的NODAL基因的研究  

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

作　　者：李念峰 方天星 倪玉芳 曾凡才 LI Nian-feng;FANG Tian-xing;NI Yu-fang;ZENG Fan-cai(Laboratory of Biochemistry and Molecular Biology,School of Basic Medical Sciences,Southwest Medical University,Luzhou 646000)

机构地区：[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

出　　处：《生物技术通报》2019年第11期240-250,共11页Biotechnology Bulletin

基　　金：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).

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。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 MDAMB-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.

关 键 词：CRISPR/Cas9 乳腺癌 基因敲除 NODAL基因 

分 类 号：Q78[生物学—分子生物学]