慢病毒介导的RNA干扰技术构建hOGG1和hMTH1基因缺陷细胞模型

Construction of hOGG1 and hMTH1 gene-deficient cell model by lentivirus-mediated RNA interference

目的:分别建立8-羟基鸟嘌呤糖苷酶1(human 8-hydroxyguanine glycosylase,hOGG1)基因和8-羟基鸟嘌呤核苷酸酶(human Mut T homlogue,hMTH1)基因缺陷细胞模型,为进一步研究两个基因的相互关系提供理想的研究平台。方法:利用慢病毒载体介导的小发夹状RNA(small hairpin RNA,shRNA)转入人胚胎肾细胞(293FT)包装慢病毒,用所得到的慢病毒感染人胚肺成纤维细胞(HFL),杀稻瘟菌素筛选稳定表达的细胞株,荧光显微镜下观察慢病毒的包装效率和感染效率,Real-time RT-PCR鉴定基因干扰效果。结果:得到了滴度较高的慢病毒,感染靶细胞后得到hOGG1基因和hMTH1基因缺陷细胞模型,hOGG1 mRNA和hMTH1mRNA表达水平分别比正常HFL细胞下降了90%和60%。结论:通过慢病毒包装技术,成功构建hOGG1基因和hMTH1基因缺陷细胞模型。

OBJECTIVE：In order to establish an ideal research platform to make further study of the relationship between the two genes,hOGG1 and hMTH1,two cell models with gene defect were established. METHODS：The lentivirus vector with shRNA targeting human 8-oxoguanine DNA glycosylase 1（hOGG1） mRNA and 8-oxoguanine nucleoside triphosphatase 1（hMTH1） mRNA were transfected into 293FT cells by liposome.Then the lentivirus supernatant was obtained and used for infecting human embryonic lung fibroblasts（HFL）.The defective cells were selected with blasticidin,and fluorescence microscope was used as a tool for observing the packaging efficiency and infection efficiency.The efficacy of gene knockout was tested by Real-time RT PCR.RESULTS：The lentivirus with high titer were obtained and the hOGG1- deficient and the hMTH1-deficient HFL cell strains were obtained after blasticidin selection.The level of hOGG1 and hMTH1 mRNA expression were decreased,90%and 60%,respectively. CONCLUSION：Lentivirus packaging technology could be mastered skillfully.hOGG 1-deficient and hMTH1-deficient HFL cell models were successfully established.