摘要
利用U6启动子转录小发卡RNA介导的RNA干涉是最近发展起来的在哺乳动物细胞中特异性抑制指定基因表达的新技术 ,已有实验证明它在小鼠畸胎瘤P19等细胞系中具有强烈的抑制基因表达的作用。本文对COS 7细胞系中U6启动子转录GFP基因的小发卡RNA介导的RNA干涉现象进行了研究 ,结果表明 :U6启动子转录的小发卡RNA具有RNA干涉作用 ,即可以在COS 7细胞中特异性地抑制含有对应序列的基因GFP的表达 ,这一结果为今后在COS 7细胞系中利用RNA干涉技术研究目的基因的功能奠定了基础。
RNA interference is a phenomenon of gene silencing directed by double stranded RNA .It can specifically inhibit gene expression by degrading mRNA efficiently and has been widely used to knockdown gene expression in Caenorhabditis elegans,Drosophila melanogaster ,etc.For mammalian cells,dsRNA directed RNAi was detected only in murine undifferentiated ES or embryonic carcinoma (EC) cells.Our previous work proved the existence of RNAi effect for reporter gene GFP and endogenous gene Oct4 in undifferentiated murine ES cells .Yet in other kinds of mammalian cells,because of the existence of interferon pathway,long dsRNA will induce the cells to shutdown global protein translation and go to apoptosis.Therefore,dsRNA longer than 30 bp cannot be used to induce specific gene knockdown effect in these cells.Elbashir et al found that in vitro synthesized small interfering RNA (siRNA)(19～23 nt)could induce potent RNAi as effective as long dsRNA without showing unspecific effect,so that the interferon pathway could be bypassed.It was shown that during RNAi process,long dsRNA was first degraded into 19～23 nt siRNA and then recruited into RISC (RNA induced silencing complex) to degrade corresponding mRNA.However,the synthesis of siRNA is expensive and the effect is transient because the knockdown effect can only be maintained for about a week.Recently,it has been shown that U6 promoter directed small hairpin RNA (shRNA) can induce potent gene knockdown effect in murine P19 Embryonic Carcinoma cell.The RNAi effect of U6 promoter driven shRNA corresponding to Green Fluorescence Protein (GFP) in COS 7 cells was checked.And it was found that the U6 promoter driven shRNA for GFP can specifically and potently knockdown the GFP's expression in COS 7 cells.The result established the feasibility of using RNAi technique directed by U6 promoter driven shRNA to study genes' function in COS 7 cell line.
基金
国家自然科学基金资助项目 (No .3 0 170 45 6)~~
