Construction And Identification Of RNA Interference Expression Vector Targeting EGFP And Effect Of Expressed NS5B On RNAi In Mammalian Cells

RNA interference is sequence-specific post-transcriptional gene silencing mediated by double stranded RNA. It can knock down the expression of homologous gene by inducing sequence-specific degradation. Since the discovery of RNAi in 1998, especially the development of vectors for production of siRNA, RNAi has been proved to be a power tool in the research of gene function and gene therapy, as an effective and specific technology that can suppress gene expression. We still do not know if the amplification of silencing signal exists in mammalian cells.pSUPER siRNA expression vector has the HI RNA promoter which can be recognized by RNA polymerase-III, as it produces a small RNA transcript lacking a poly-adenosine tail and has a well-defined start of transcription and a termination signal consisting of five thymidines in a row. The resulting transcript is predicted to fold back on itself to form a 19-base pairstem-loop structure and then be processed as siRNA, resembling that of synthesis siRNA, so that it can suppress the expression of target gene.The reporter gene, enhanced green fluorescence protein (EGFP) used in our research has no toxic effects on mammalian cells. It can be as a good marker of gene expression, alone or fusioned with other genes. The expression of the gene can be easily detected and be supervised real time. So this reporter gene is widely used in the research on gene expression of mammalian cells. We constructed three pSUPER siRNA expression vectors targeting EGFP mRNA. After enzyme analysis and DNA sequencing confirmation, the recombinants were named pSUPER-R237, pSUPER-R304 and pSUPER-R447. pIRESE2-EGFP was co-transfected into COS-7 cells transiently with pSUPER-R237, pSUPER-R304 and pSUPER-R447 respectively. Fluorescence microscopical examination was performed 48h after transfection to investigate the expression of EGFP. The results indicated that the three recombinant pSUPER expression vectors targeting different sites of EGFP gene could suppress expression of EGFP gene significantly.pSUPER-Rs was also co-transfected into COS-7 cells with pEGFP-N3-14S plasmid, which inserted with a single chain Fv gene against HBsAg fused with EGFP at the C-terminus. We investigated the suppression effect of pSUPER-R447 vector on the recombinant EGFP fused gene. The results indicated that siRNA targeting the coding sequence of EGFP could also suppress the expression of the gene fused with EGFP.In C. elegans, only few molecules of dsRNA can induce intense RNAi effec. Accounting for this phenomenon requires a system to amplify the signal. RNA-dependent RNA polymerase (RdRp) could be involved inamplifying the RNAi signal in plants and C. elegans. At present, similar RNAi amplification system has not been found in mammals. Moreover, no RdRp protein or homologs have been detected and isolated. NS5B protein is nonstructural protein five of HCV (Hepatitis C virus), which is an RNA-dependent RNA polymerase responsible for the viral RNA synthesis. It can copy RNA template by its primer-dependent RdRp activity.In this study, we try to express exogenous RdRp (NS5B of HCV) in mammals. We want to know whether the RdRp-directed amplification mechanism could be constructed with exogenous RdRp as that in plants or C. elegans.In our research, template oligos were designed and inserted into pSUPER. Then we constructed a recombinant pSUPER expression vector, pSUPER-311, which could express primer RNAs complementary to the 3' noncoding sequence of EGFP gene. If the RdRp-directed amplification mechanism be established, dsRNA would be synthesized with primer RNAs as primer and the target mRNA as template. The nascent dsRNA could be processed into siRNAs by Dicer. Then the siRNAs would be involved into RNAi pathway and the expression of EGFP coding sequence would be suppressed.pIRES2-EGFP-NS5B expression vector was also constructed which can express EGFP and NS5B simultaneity. The recombinant expression vector and pSUPER-311 were co-transfected transiently into COS-7 cells and the suppression effect of EGFP was assayed by fluorescence microscope.For the convenience of screening, we constructed recombinant expression vectors that can express siRNAs and NS5B simultaneity. The recombinant expression vectors are pCDNA3.1(-)-H1-311,...