利用CRISPR/Cas9系统构建HepG2细胞THRAP3基因敲除细胞系

THRAP3 gene knockout in HepG2 cell line using CRISPR/Cas9 system

目的使用CRISPR/Cas9基因编辑系统敲除人类肝癌细胞HepG2中的甲状腺激素受体相关蛋白3(THRAP3)基因,构建THRAP3基因敲除细胞系。方法根据CRISPR/Cas9靶点设计规则,设计特异性靶向THRAP3基因第3个外显子相关序列的多条小向导RNA,通过T7核酸内切酶筛选出切割效率最高的载体,构建pSpCas9-THRAP3真核重组表达质粒。测序鉴定后,将重组质粒转染至HepG2细胞中,用嘌呤霉素(puromycin)抗性筛选细胞株,挑取单克隆,再用PCR、测序等方法鉴定细胞基因型,免疫印迹方法鉴定细胞THRAP3敲除效果。使用高通量测序的方法进行基因表达分析。用流式细胞仪检测基因敲除对细胞周期的影响,CCK-8试剂盒检测细胞增殖。结果成功构建pSpCas9-THRAP3真核重组表达质粒,筛选出特异性敲除THRAP3基因的细胞系,验证了THRAP3敲除对细胞周期以及细胞增殖的影响。结论利用CRISPR/Cas9基因编辑技术成功构建并获得了THRAP3基因敲除细胞系并初步探究了THRAP3基因功能。

Objective To knockout the thyroid hormone receptor associated protein 3(THRAP3)gene in human hepatocellular carcinoma HepG2 cells using the CRISPR/Cas9 gene-editing system.Methods According to the protocol of the CRISPR/Cas9 system,the group of sgRNAs that specifically targeted the third exon of THRAP3 gene was designed before those with the highest efficiency were tested by T7 EI filter.After sequencing,the plasmid was transfected into HepG2 cells,before cell lines that could stably knockout THRAP3 were screened under the stress of puromycin.The gene knockout efficiency was detected by PCR and Western blotting.Gene expression was analyzed by using high-throughput sequencing.The effect of gene tapping on cell cycle and cell proliferation was detected by flow cytometry and CCK8.Results A pSpCas9-THRAP3 eukaryotic recombinant expression plasmid was constructed.Cell lines that could specifically knockout THRAP3 were selected.Conclusion CRISPR/Cas9 gene editing technology can be used to construct and obtain THRAP3 gene knockout cell lines.