以miRNA为基础的RNA干扰线粒体相关基因TFAM和POLG的研究

miRNA-based RNAi mitochondria-related genes TFAM and POLG

摘要: 目的通过慢病毒介导的RNA干扰技术干扰线粒体相关基因TFAM和POLG,为研究线粒体和干细胞提供资料。方法选取线粒体相关因子TFAM和POLG,分别设计三条干扰序列,构建以miRNA为基础的RNA干扰病毒载体。在293FT细胞中包装病毒液,并转染原代成纤维细胞,通过定量PCR和western blot检测干扰效率。结果两个基因的1号序列都能有效干扰相应基因表达,POLG下调了75%,而TFAM下调了85%。结论慢病毒介导的miRNA为基础的干扰病毒载体成功干扰了TFAM和POLG基因的表达。

Abstract: Objective To provide the data for mitochondria and stem cells by studying lentivirus-mediated RNAi mitochondria-related genes TFAM and POLG. Methods Mitochondria-related genes TFAM or POLG were selected.Three shRNAs were designed to construct RNAi lentivirus vectors based on miRNA.Lentivirus plasmids were packaged into 293FT cells,transfected into primary fibroblasts,and efficiency of RNAi was detected by quantitative PCR and Western blot,respectively. Results POLG-shRNA-1 decreased the TFAM and POLG expression of 75% and 85%,respectively. Conclusion Lentivirus-mediated RNAi vector can interfere the expression of miRNA-based POLG and TFAM.