Inhibition Effect Of RNAi On CTCF Expression By Adeno-associated Virus Vector

RNAi (RNA interference) is a process of sequence-specific, post-transcriptional gene silencing in animals and plants initiated by double-stranded RNA that is homologous to the silenced gene and has proven as a novel approach to suppress gene expression specifically. Adneo-associated virus vector is an efficient delivery system to introduce small DNA fragment into cultured mammalian cells safely. CTCF is an exceptionally conserved transcription factor that is widely expressed in eukaryotics. And it interacts with multiple gene by usage of 11 zinc fingers and is related to multiple functions.It's the first study which aimed to repress CTCF expression in somatic cell lines by siRNA interference and carry out the subsequent global analysis of CTCF-regulated gene candidates.In this study, we first designed target siRNA aimed at CTCF mRNA and selected two targets. To assess the packing recombined adneo-associated virus plasmid, a shRNA expression vector was constructed with H1 promoter and the selected targets was cloned in it. Recombined adneo-associated virus infected the HeLa cells and the monoclonal cell that can express shRNA stably was selected. We detected the CTCF expression of obtained monoclonal cell by real-time PCR and western blotting. It was demonstrated that the CTCF mRNA expression was knockekd down 76% compared with normal HeLa cells by real-time PCR with GAPDH as an inner reference. It was also demonstrated that the CTCF protein expression was knocked down about 70% contrasted with normal HeLa cells by western blotting with α -actinin as an inner reference . It seems to be some correlation between repression of CTCF mRNA, protein and inhibition of cell growth.Moreover, in this study we exploited efficient condition of transfection by means of liposome DC-chol/DOPE. We prepared liposome in the lab and found the optimum...