特异性shRNA干扰人慢病毒载体抑制U251细胞株Chk1、Chk2基因的表达

Inhibition of Chk1 and Chk2 gene expression in U251 cell line using Lentivirus-mediated shRNA

目的构建针对细胞周期检测点激酶1和2(Chk1和Chk2)基因的短发夹RNA(shRNA)表达载体,包装成慢病毒,建立稳定转染的细胞株,为探讨抑制Chk1和Chk2基因表达对脑胶质瘤细胞生物学行为调控的研究奠定基础。方法根据GenBank数据库提供的Chk1和Chk2基因核苷酸序列,选择设计2条能转录短发夹RNA(shRNA)的DNA序列,命名为Chk1-shRNA和Chk2-shRNA,同时设计1条非特异性序列作为阴性对照,命名为blank-shRNA。并与pLKO.1-TRC质粒载体连接,转化感受态大肠杆菌,挑取阳性克隆,抽取重组质粒,使用限制性内切酶EcoRⅠ、NcoⅠ酶切电泳,DNA测序鉴定,包装慢病毒。3组重组表达慢病毒载体转染胶质瘤细胞系U251,用嘌呤霉素筛选后挑选单克隆并扩增获得稳定株。逆转录酶-聚合酶连反应(RT-PCR)和Western blot分别在mRNA和蛋白水平上检测Chk1和Chk2的表达。结果重组质粒成功转化感受态大肠杆菌,经酶切琼脂糖凝胶电泳分析,结果表明寡核苷酸成功插入到预计位点,经测序鉴定,序列完全正确。嘌呤霉素对U251细胞的筛选浓度为4ug/ml,筛选出稳定转染三种质粒的U251细胞,Chk1-shRNA和Chk2-shRNA组细胞各自Chk1和Chk2的mRNA和蛋白表达水平明显低于blank-shRNA组。结论成功构建了针对Chk1和Chk2基因的shRNA慢病毒表达载体,转染后可抑制ChK1和Chk2基因的表达,为进一步研究Chk1和Chk2基因在脑胶质瘤细胞中的作用奠定了基础。

Objective To construct eukaryotic expression vector of RNA interference for Chk1 and Chk2 gene, and to screen the stably transfected cell clones. Methods specific Genomic sequences of Chk1 and Chk2 gene were retrieved from Genbank and cDNA were designed and synthesized, and one scrambled shRNA sequences served as negative control. The cDNA were synthesized and inserted into plasmid pLKO. 1. Recombinant vectors were transformed into competent E. coli. The positive clones were selected and recombinant plasmids were extracted. The plasmids were digested with EcoR I and Nco I , then loaded in agarose gel electrophoresis. The three shRNA vectors were transfected into U251, the stably transfected cell clones were obtained after being screened with puromycin. Western blotting and Reverse transcriptase-polymerase chain reaction （RT-PCR） were performed to examine the inhibitory effect at the RNA level and protein level. Results The stably transfected pLKO. 1-TRC-Chk1-shRNA and pLKO. 1-TRC-Chk2-shRNA cell clones were significantly down-regulated by siRNA as validated by Western blotting and RT-PCR. Conclusion RNA interfering （RNAi） mediated by the shRNA expressing vectors can significantly down-regulate the expression of Chk1 and Chk2 in glioma cell line U251. The stably transfected cell clones were obtained for further study.