靶向HIF-1α基因的CRISPR/Cas9基因敲除质粒的构建与鉴定*
Construction and indentification of CRISPR/Cas9 plasmid targeting HIF-1α gene

目的:构建靶向低氧诱导因子(HIF)-1α基因的CRISPR/Cas9基因敲除质粒,并体外鉴定其敲除效果。方法:设计靶向HIF-1α基因的gRNA序列,将其插入到CRISPR/Cas9质粒骨架载体pCAG-T7中,转化后挑取克隆,进行测序验证。将构建好的重组质粒体外转染人肝癌细胞HepG2和SK-Hep-1,利用嘌呤霉素进行筛选并扩大培养获得HIF-1α基因敲除混合克隆细胞,应用Western blot技术检测未转染细胞和混合克隆细胞中HIF-1α蛋白的表达情况。结果:经测序验证,成功构建了靶向HIF-1α的重组质粒pCAG-T7-HIF-1α-gRNA,经Western blot验证,转染该重组质粒后人肝癌细胞株HepG2和SK-Hep-1经CoCl2诱导HIF-1α蛋白表达明显减弱。结论:成功构建了靶向HIF-1α基因的CRISPR/Cas9基因敲除质粒载体。
Aim: To construct CRISPR/Cas9 knock-out plasmid targeting HIF-1α gene, and identify its knock-out effect in vitro.Methods: The gRNA sequences targeting HIF-1α gene were designed,then were inserted into CRISPR/Cas9 plasmid skeleton vector pCAG-T7,which was transformed into competent DH5α.The single clone was picked up and validated via sequencing. Then the constructed recombinant vector pCAG-T7-HIF-1α-gRNA was transfected into HepG2 and SK-Hep-1, and the HIF-1α gene knock-out mixed colony cell were selected using puromycin. Subsequently, the expression level of HIF-1α in the HIF-1α gene knock-out mixed colony cell lines was detected by Western blot.Results: Through the sequencing validation, the plasmids targeting HIF-1α gene was successfully constructed. The expression levels of HIF-1α in HepG2 and SK-Hep-1 transfected with the recombinant vector were significantly reduced.Conclusion: CRISPR/Cas9 plasmid targeting HIF-1α gene has been successfully constructed