Synthetization And Appraisal Of Lentiviral ShRNA Vector Carrying Human ATP2A3Gene

Objectives To synthetize and appraise lentiviral shRNA vector carrying humanATP2A3gene to provide materials for further studying of mechanism whichsalinomycin resisted cancer stem cells through regulating and controlling ATP2A3gene.Methods1. Synthetized four lentiviral shRNA vectors carrying human ATP2A3gene: firstly, interference sequence of ATP2A3was connected to pGMLV-SC1RNAilentiviral vector; secondly, connection product was switched to competent cells inBacteria; lastly, the growing monoclonal bacterial colony was identified by DNAsequencing. The correct clon by DNA sequencing was the lentiviral RNAi vectorcarrying target gene ATP2A3.2. Packaged lentivirus and measured titer: highlypurified and non-endotoxic lentiviral shRNA vector and auxiliary vector wereextracted, and then transfected to293T cells through HG transgene reagent, thelentiviral-rich supernatants was collected after promoting transfection, changing thesolution and breeding. High titer of lentiviral was obtained and measured bycondensing the supernatants.3. Testted effect of lentiviral acting on ATP2A3: madelentiviral and NC-lentiviral infect pc-3cells, and evaluated jamming effect oflentiviral vector acting on target gene by Western blot and RT-PCR.Results1. Four lentiviral vectors were identified by DNA sequencing that allDNA sequences did not contain mutation site. It proved that construction of plasmidwas successful.2. After transfecting lentiviral vector plasmid to293T cells, greenfluorescence was obviously observed. To Package lentivirus particles in293T cells, after purifying, high titer of pGMLV-SC1-ATP2A3plasmid was received eventually.3. Target cells were tested by western blot and RT-PCR that target gene ATP2A3infour lentiviral vector were certainly down-regulated and the ATP2A3-shRNA4wasthe most obvious.Conclusion1. Lentiviral shRNA vector carrying human ATP2A3gene could besuccessfully synthetized by RNAi technology.2. The synthetized interference vectorcould be successfully packaged by lentiviral, and high titer of pGMLV-SC1-ATP2A3plasmid could be received eventually.3. The testing results by RT-PCR and WBindicated that ATP2A3-shRNA4owned the best silence effects of four target spots inATP2A3gene. And PC-3cell lines affected by ATP2A3-shRNA4could be act asexperiment materials uesing for further mechanism study of salinomycin resisttingcancer stem cells through regulating and controlling ATP2A3gene.