Construction of SiRNA Lentivirus Vector and the Influence on Sosp9607

To construct a lentiviral vector of RNA interference (RNAi) of human cyclin E gene, the overexpression vector pcDNA—cyclin E was confirmed in our previous study. 4 sequence of siRNA targeting human cyclin E gene was designed,synthesized and co-transfected into 293T cells to screen the effective one. The complementary DNA containing both sense and antisense Oligo DNA of the effective sequence siRNA—464 was cloned into the pLKO.1 vector. The resulting lentiviral vector containing cyclin E shRNA was named pLKO—CE and it was confirmed by restriction endonuclease analysis and sequencing. 293T cells were co-transfected with lentiviral vector pLKO—CE,VSV—G and Δ8.9. All virus stocks were produced by calcium phosphate mediated transfection,and the supernatant of transfected 293T cells was collected to obtain the recombinant lentivirus. The interference effective of virus was tested according to the expression level of cyclin E on Sosp9607 cells.It is resulted: that PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of cyclin E producing shRNA was constructed successfully,and which can inhibit the expression of cyclin E molecule in Sosp9607 cell effectively. It is concluded that lentivirus RNAi vector of cyclin E is constructed successfully.