利用CRISPR/Cas9建立RBBP4低表达的HepG2,LO2稳定细胞系  

The establishment of HepG2 and LO2 stable cell lines with low RBBP4 expression using CRISPR/Cas9

作　　者：杨丽丽 黄常新[1] 李永强[1] 王聪洁 沈靖 陈艺丹 张嗣玉 Yang Lili

机构地区：[1]杭州师范大学附属医院,310000 [2]临安市人民医院,311399 [3]杭州市余杭区二院,311121 [4]杭州市肿瘤医院,310005

出　　处：《浙江临床医学》2022年第12期1742-1744,共3页Zhejiang Clinical Medical Journal

基　　金：浙江省重大科技项目(2017C03053)。

摘　　要：目的利用CRISPR/Cas9在肝癌细胞HepG2及正常肝组织细胞(LO2)中敲低RBBP4 eGFP报告基因,构建RBBP4低表达的HepG2及LO2稳定细胞系.方法基于CRISPR/Cas9系统原理,设计靶向细胞RBBP4基因第1个外显子的小向导RNA(sgRNA),构建具有左右同源臂和P2A-eGFP供体质粒的干扰质粒;将测序鉴定正确的重组质粒分别转染至HepG2及LO2细胞中,通过嘌呤霉素筛选细胞系,提取RNA和蛋白,分别通过QPCR和蛋白质印迹分析检测RBBP4敲低对细胞系的影响.结果QPCR及和蛋白质印迹分析结果提示筛选的HepG2及LO2细胞系中RBBP4基因及蛋白表达减低(P<0.05).结论通过CRISPR/Cas9系统成功构建了RBBP4稳定敲低的HepG2及LO2细胞系.Objective To use the CRISPR/Cas9 technology to knock down the RBBP4 enhanced green fluorescent protein(eGFP)reporter gene in hepatocarcinoma cells HepG2 and normal liver tissue cells(LO2)to construct a stable cell line of HepG2 and LO2 with low expression of RBBP4.Methods Based on the principle of CRISPR/cas9 system,a small guide RNA(sgRNA)targeting the first exon of rbbp4 gene was designed,and an interference plasmid with left and right homologous arms and P2a EGFP donor plasmid was constructed.The recombinant plasmids identified by sequencing were transfected into HepG2 and LO2 cells respectively.The cell lines were screened by puromycin,and the RNA and protein were extracted.The effects of RBBP4knockdown on the cell lines were detected by QPCR and Western blot analysis.Results The results of QPCR and Western blotting indicated that the expression of RBBP4 gene and protein was reduced in the selected HepG2 and LO2 cell lines(P<0.05).Conclusion The HepG2 and LO2 cell lines with stable knockdown of RBBP4 are successfully constructed through the CRISPR/Cas9 system.

关 键 词：视网膜母细胞瘤结合蛋白4 CRISPR/Cas9 系统HepG2细胞 LO2细胞 

分 类 号：Q78[生物学—分子生物学]