SRSF9基因敲除胶质母细胞瘤细胞株基因回补及其功能研究

Gene rescue and function study of SRSF9 knockout glioma cell line

目的在CRISPR/Cas9敲除SRSF9基因的胶质母细胞瘤U87单克隆株中进行SRSF9基因回补并进行回补功能研究。方法构建SRSF9-WT过表达载体并同义突变sgRNA1靶序列上的5个碱基构建SRSF9-sgMu过表达载体并包装成慢病毒,感染U87 SRSF9基因敲除单克隆细胞,构建SRSF9基因回补细胞株。利用Western blot和免疫荧光实验验证回补蛋白的定位,利用增殖和肿瘤干细胞成球实验验证回补基因的拯救功能。结果成功构建SRSF9-WT和SRSF9-sgMu过表达载体并包装成慢病毒。Western blot和免疫荧光实验验证回补成功,且回补的SRSF9蛋白优势表达于细胞核。增殖和肿瘤干细胞成球实验证明回补的SRSF9可以拯救其功能(P<0.05)。结论SRSF9-sgMu成功在SRSF9基因敲除的U87单克隆细胞中回补SRSF9并拯救增殖和肿瘤干细胞成球功能。

Objective To rescue SRSF9 gene in human glioma U87 SRSF9 knockout monoclonal cell line using CRISPR/Cas9 system and to research its function.Methods SRSF9-WT and SRSF9-sgMu overexpression vector which was synonymous mutated 5 bases on site of SRSF9-WT were conducted and packaged into lentivirus.U87 SRSF9 knockout monoclonal cell lines were infected with lentivirus to construct SRSF9 gene rescue cell lines.Western blot and immunofluorescence assays were used to detect the localization of rescued protein,proliferation experiment and tumor stem cell sphere formation assay were used to detect the rescue functions of rescued gene.Results SRSF9-WT and SRSF9-sgMu overexpression vector were successfully constructed and packaged into lentivirus.Western blot and immunofluorescence assays confirmed that the rescue was successful and therescued SRSF9 protein was predominantly expressed in nucleus.Theresultsofproliferation and tumor stem cell sphere formation assays showed that the rescued SRSF9 could also rescue its function(P<0.05).Conclusion SRSF9-sgMu can successfully rescue the expression of SRSR9 and its function of proliferation and tumor stem cell sphere formation in human glioma U87 SRSF9 knockout monoclonal cell line.