慢病毒介导构建661W细胞mito-OGG1过表达和OGG1敲减模型

Construction of lentivirus-mediated mito-OGG1 gene overexpression and OGG1 gene knockdown model in 661W cells

目的构建和鉴定慢病毒介导的小鼠线粒体靶向8-羟基脱氧鸟嘌呤DNA糖苷酶1(mito-OGG1)基因在661W细胞中的过表达,以及慢病毒介导的短发夹RNA(shRNA)下调OGG1基因在661W细胞中的表达。方法构建目的质粒pLenti-EF1a-EGFP-P2A-Puro-CMV-Mito-OGG1-3Flag(pLenti-OGG1-GFP)和pLKD-CMV-G&PR-U6-shRNA(pLKD-shRNA),利用第2代慢病毒包装系统和293T细胞获得绿色荧光蛋白(GFP)标记的目的慢病毒载体,利用293T细胞进行病毒滴度检测,荧光显微镜计数法检测不同感染复数(MOI)梯度下661W细胞的转染效率和生存情况,并确定最适MOI,利用不同浓度梯度嘌呤霉素作用于661W细胞以筛选出嘌呤霉素最低使用浓度,并以此浓度筛选出稳定转染细胞株,免疫荧光检测筛选后细胞转染效率并进行细胞色素C氧化酶Ⅳ(COXⅣ)-OGG1共定位鉴定,采用荧光定量PCR(QPCR)和Western blot法分别检测OGG1基因的mRNA和蛋白相对表达水平。结果测序结果显示,过表达质粒中插入的序列与基因库中小鼠OGG1(NM_010957.4)基因序列一致,包装纯化后的慢病毒原液滴度为2.0×10^7~6.0×10^7 TU/ml,661W细胞最适MOI为40,4.0 μg/ml嘌呤霉素成功筛选出稳转株,筛选后转染效率达100%。免疫荧光染色结果显示,OGG1与COXⅣ成功共定位。空白对照组、OGG1组、过表达对照组、shRNA组和低表达对照组OGG1 mRNA相对表达量分别为1.000±0.000、41.581±12.206、0.888±0.056、0.239±0.121和1.081±0.083,OGG1蛋白的相对表达量分别为1.029±0.153、1.657±0.237、0.752±0.143、0.471±0.149和1.036±0.185,各组间总体比较差异均有统计学意义(F=44.654、30.948,均P<0.05),其中OGG1组的OGG1 mRNA和蛋白相对表达量均较过表达对照组明显升高,shRNA组的OGG1 mRNA和蛋白相对表达水平均较低表达对照组明显降低,差异均有统计学意义(均P<0.05)。结论本实验成功构建661W细胞mito-OGG1过表达和OGG1敲减模型,为下一步研究提供了实验基础。

Objective To construct and authenticate the lentiviral-mediated overexpression of mouse mitochondrial-targeted-8-oxoguanine DNA-glycosylase 1 (mito-OGG1) gene and the lentiviral-mediated short hairpin RNA (shRNA) down-regulation of OGG1 gene expression model in 661W cells. Methods Constructed the target plasmids, including pLenti-EF1a-EGFP-P2A-Puro-CMV-Mito-OGG1-3Flag (pLenti-OGG1-GFP) and pLKD-CMV-G&PR-U6-shRNA (pLKD-shRNA).293T cells were used to obtain green fluorescent protein (GFP)-tagged lentiviral vector of interest by using a second generation lentivirus packaging system.293T cells were also used for the virus titer estimation.The multiplicity of infection (MOI) of 661W cells was detected by fluorescence microscopy.A stable transfected cell line was screened by puromycin.Immunofluorescence was used to detect transfection efficiency and cytochrome C oxidase Ⅳ(COXⅣ)-OGG1 co-localization.OGG1 mRNA and protein expression levels were detected by real-time qantitative PCR (QPCR) and Western blot. Results Sequencing results showed that the inserted sequence in the over-expression plasmid was consistent with the mouse OGG1 (NM_010957.4) gene sequence in the gene library.The original lentiviral titer after packaging and purification was between 2.0×107 to 6.0×107 TU/ml.The optimal MOI of 661W cells was 40, and puromycin with a concentration of 4.0 μg/ml successfully screened stable transformation.The transfection efficiency was up to 100% after screening.Immunofluorescence demonstrated successful co-localization of OGG1 and COXⅣ.The relative expression levels of OGG1 mRNA in the blank control group, OGG1 group, overexpression control group, shRNA group and low expression control group were 1.000±0.000, 41.581±12.206, 0.888±0.056, 0.239±0.121 and 1.081±0.083, and the relative expression levels of OGG1 protein were 1.029±0.153, 1.657±0.237, 0.752±0.143, 0.471±0.149 and 1.036±0.185, respectively, with significant differences between them (F=44.654, 30.948;both at P<0.05), the relative expression levels of OGG1 mRNA and protein in the OGG1 group were significantly higher than those in the overexpression control group, the relative expression levels of OGG1 mRNA and protein in the shRNA group were significantly lower than those in the lower expression control group, with significant differences between them (all at P<0.05). Conclusions The mito-OGG1 overexpression and OGG1 knockdown models of 661W cells are successfully constructed, which provides the preliminary experimental basis for follow-up study.