The Establishment Of The Plasmid-based Rna Interference Method, And Apoptosis Related Gene Hsd11 Function

There are two parts in this thesis. In the first part, the plasmid-based RNA interference technology was established, and in the second part, the function of HSD11 gene was studied.Although RNA interference phenomenon was discovered only years ago, it has been developed as a key method to study gene function. During the past several years, it has been widely used in studying the function of genes in nematode, fly and mammalians. RNA interference in mammalian cells was aroused by siRNA as long as 19bp to 21bp, therefore chemical synthesized siRNA was the prevalent way to study gene function in mammalian cells. It is impossible to obtain cells, however, in which the target gene was persistently inhibited with siRNA, Therefore it is potential to use plasmid-based RNA interference technology to study gene function.In cells, snRNAs (small nuclear RNAs) were transcribed by RNA polymerase III, which was universally expressed in all cells. The initiator site and terminator site of RNA polymerase III are very simple and the transcriptional products are small RNA as long as tens to hundreds nucleotide, so it is properly to transcribe small RNA for RNA interference. The promoter that RNA polymerase III recognize are U6 promoter or H1 promoter, therefore the key step of plasmid-based RNA interference technology is cloning the RNA interference target sequence to downstream of U6 or H1 promoters.In this study, EGFP was firstly used as target gene to establish RNA interference technology. Three target sites were cloned into RNA interference vector psilencer-1.0-U6, and then the plasmid were cotransfected with pEGFP-N1 into Hela cells, respectively. The results of fluorescence microscopy, FACS and Western blot indicated that the expression level of EGFP was effectively inhibited by the RNA interference plasmids, which proved that RNA interference plasmids could inhibited the expression level of exogenous gene cotransfected. In the following work, HSD11 gene was used as target gene to study the efficiency of RNA interference plasmid to endogenous gene. Six interference target sites were screened and cloned into psilencer-2.1-U6-neo or psilencer-1.0-U6, respectively. The results of RT-PCR, immunofluorescence microscopy, and Western blot indicated that the expression level of endogenous HSD11 was effectively inhibited by the RNA interference plasmids. Several issues affecting the RNA interference efficiency such as screening the...