基于CRISPR/Cas9系统构建FOXA2表达示踪体系  

Construction of FOXA2 Expression Tracer System Based on CRISPR/Cas9 System

作　　者：程浩杰 张振旺 谭桂湘[1] 刘昱翔 卜会铜 谭拥军[1] CHENG Hao-jie;ZHANG Zhen-wang;TAN Gui-xiang;LIU Yu-xiang;PIAO Hui-tong;TAN Yong-jun(College of Biology, Hunan University, Changsha 410082)

机构地区：[1]湖南大学生物学院,长沙410082

出　　处：《生物技术通报》2018年第5期80-86,共7页Biotechnology Bulletin

基　　金：国家自然科学基金资助项目(81472718)

摘　　要：利用CRISPR/Cas9系统在基因FOXA2蛋白质编码区后插入绿色荧光蛋白核酸序列,实现对FOXA2基因开启和关闭的示踪。通过靶向序列选择、Cas9靶向质粒及Donor片段的克隆、细胞转染并进行流式细胞荧光分选、阳性细胞有限稀释、富集培养及单克隆细胞的鉴定和转录翻译水平验证等5个方面,进行实验研究。在编辑的乳腺肿瘤细胞MCF-7中,绿色荧光蛋白有效表达;乳腺癌细胞内绿色荧光蛋白的表达水平同FOXA2蛋白的表达水平正相关,且经肿瘤细胞上皮间质转化进程诱导因子EGF的诱导,绿色荧光蛋白和FOXA2表达水平同步降低。方法学上CRISPR/Cas9靶向FOXA2并利用细胞内同源性定向修复插入绿色荧光蛋白序列成功;利用报告基因蛋白表达水平示踪目的基因的开启、关闭及表达量高低结果准确、方法简单,效果明显。This study enabled the tracing of FOXA2 gene on and off by using CRISPR/Cas9 system to insert a green fluorescent protein（GFP）nucleic acid sequence following the FOXA2 protein coding region. The study was carried out from the following five aspects ：targetingsequence selection,cloning of Cas9-targeting plasmids and donor fragment,cells transfection and flow cytometry fluorescence sorting,limited dilution of positive cells,as well as enrichment culture and identification,transcription and translation level verification of monoclonalcells. The results showed that the expression of GFP was positive in the edited breast cancer cell line MCF-7. The expression level of GFP inbreast cancer cells was positively correlated with the expression level of FOXA2 protein,and the expression of GFP and FOXA2 decreasedsynchronously after the induction by EGF,an inducing factor in the process of tumor cells epithelial-mesenchymal transition（EMT）. Theresults indicate that CRISPR/Cas9 targeting FOXA2 and using intracellular homology directed repair（HDR）to insert GFP sequence aresuccess. Using the expression level of reporter gene protein for tracing the target gene on and off as well as its expression is significantly accurateand convenient.

关 键 词：CRISPR/Cas9 基因编辑 FOXA2 示踪 乳腺癌 MCF-7 上皮间质转化 

分 类 号：Q78[生物学—分子生物学]