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目的运用CRISPR/Cas9基因编辑技术,在人恶性横纹肌样瘤细胞株G401中敲除p21基因。方法通过反转录定量PCR(RT-q PCR)及Western blot检测各瘤细胞株中p21的表达,针对p21基因作用的功能域,设计了靶向人p21基因第3个外显子的向导RNA(sg RNA),克隆入lenti CRISPR v2载体。将测序及酶切鉴定正确的重组质粒在293T工具细胞中制备慢病毒颗粒并感染G401细胞,使用嘌呤霉素进行阳性细胞筛选,显微镜下挑取单克隆细胞团并继续培养获得G401单克隆细胞株。提取单克隆细胞株RNA及蛋白,利用RT-q PCR及Western blot方法检测细胞株中p21的敲除效果。结果 p21在人横纹肌样瘤细胞中高表达。成功构建靶向p21基因的lenti CRISPRv2-sg RNA重组慢病毒质粒。与对照组相比,筛选得到的G401亚克隆细胞系中p21蛋白表达缺失。结论针对难转染的G401细胞,应用CRISPR/Cas9系统成功构建了p21基因敲除的稳定株,为后续深入研究p21在人恶性横纹肌样瘤中的作用机制奠定了基础。
Objective To knock out the p21 gene in human malignant rhabdomyosarcoma cell line G401 by using CRISPR / Cas9 gene editing technology. Methods The expression of p21 in each tumor cell line was detected by reverse transcription-polymerase chain reaction (RT-q PCR) and Western blot. For the functional domain of p21 gene, a guide RNA targeting the third exon of human p21 gene was designed (sg RNA) and cloned into lenti CRISPR v2 vector. The correct recombinant plasmids were identified by sequencing and restriction enzyme digestion. Lentiviral particles were prepared in 293T tool cells and infected with G401 cells. Puromycin was used to select positive cells. Monoclonal colonies were picked under the microscope and cultured continuously to obtain G401 monoclonal cell lines . The RNA and protein of monoclonal cell lines were extracted and the knockdown of p21 in cell lines was detected by RT-q PCR and Western blot. Results p21 was highly expressed in human rhabdomyosarcoma cells. The lenti CRISPRv2-sg RNA recombinant lentiviral plasmid targeting the p21 gene was successfully constructed. Compared with the control group, the p21 protein expression in the screened G401 subclone cell line was absent. Conclusion The stable knock-out p21 gene was successfully constructed by using CRISPR / Cas9 system in G401 cells, which lays the foundation for further study on the mechanism of p21 in human rhabdomyosarcoma.