Construction And Confirmation Of RNA Interference Vectors Based On Different Promoters

RNA interference (RNAi) is a post-transcriptional gene silencing induced by the introduction of double-stranded RNA(dsRNA) which triggers the degradation of a homologous mRNA.The dsRNAs transfer into cells, RNAi destroys the target RNA which shares sequences homologous with dsRNA and inhibits the target gene expression effectively.To develop a stable RNA interference system, we sought to construct inducible targeting RED gene vectors drived by different types of promoters.The constructed vectors were transfected into HeLa cells to prove the stability of the system and provide a technological method for studying tissue-specific RNA interference and a new idea for researching on transgene wool-using animals.Chapter1Construction of RNA interference vectorsThe CMV promoter was cloned from pcDNA3.1(+/-) plasmid, K5pomoter was cloned from pGL3-K5plasmid, KAP6.1promoter was cloned from sheep cDNA and UHS promoter was cloned from mouse genome.Connected the PCR products and pMD19-T simple vectors to form middle vectors. Digested the middle vectors and pGenesil-1by BamHI&EcoRI and purified the purpose fragments. Connected the purpose fragments and transformed the connect products into E.coli DH5α.The lengths of these promoters were660bp,870bp,1052bp and580bp, respectively. By blast the sequences in GenBank showed the transition vectors were correct. The shRNA fragments were cloned from three different shRNA expression vectors. Then digested the shRNA fragments and modified pGenesil-1vectors which U6promoter was replaced by CMV, K5, KAP6.1and UHS promoters by HindⅢ and BamHI. Then inserted the purpose shRNA fragments into the modified pGenesil-1fragments. The lengths of these shRNA fragments were115bp,116bp and117bp, respectively. By blast the sequences in GenBank showed the PCR products sequences were correct. The fifteen RNA interference vectors targeting RED gene were constructed successfully. Chapter2Confirmation of the RNAi vectorsIn order to comfirm the inhibition efficiency of fifteen RNAi vectors, the RFP expression was observed. Divided fifteen constructed vectors into five groups to transfect into HeLa cells, every group repeated four times. At the same time transfected CMV-Red plasmids into HeLa cells. Fourty-eight hours after transfection, the RFP expression levels were observed by Fluorescent microscope. RT-PCR and Real-time PCR techniques were used to investigate the efficiency of inhibition. The results showed that compared with the control group, both of the fifteen interference vectors reacted in HeLa cells, the highest RNAi efficiency was up to90%.K5-driven RNAi vectors and CMV-Red plasmids were choosed to co-transfected into sheep fetus fibroblast which express RFP stably. The results implied that after fourty-eight hours transfection, the green fluorescent was noticed and the red fluorescent was decreased. All the details showed that the RNAi vectors structures were correct, they both drived the shRNA expression and inhibited the RFP expression.