Preparation the recombinant retrovirus expressing siRNA of Pokemon gene and the suppression on Hela cell proliferation

Objective To prepare the recombinant retrovirus expressing siRNA of Pokemon and determine the suppressing effects of the retrovirus on Hela cell proliferation.Methods Four sequences corresponding to the human Pokemon genes were designed on Ambion'S Web.The 4 complementary DNA fragments were synthesized and cloned into the sites of BamHⅠ and HindⅢ of the pSihncer5.1-H1 retovirus vector for constructing the recombinant retrovirus plasmid.Hela cell lines that over-express Pokemon gene were determined by RT-PCR.Then these plasmids which expressed the 4 different siRNAs were used to directly transfect Hela cells.After puromycin screening,the cells which were stably transfected with these plasmids were cultured.The silencing effects of Pokemon gene in these four groups were determined by real time RT-PCR assay.At the same time,the proliferation conditions of these four group cells were recorded by MTT assay.Then the most effective plasmid which could silence Pokemon gene and suppress Hela cells proliferation was chosen to prepare the recombinant retrovirus expressing siRNA of Pokemon gene.At the end,the retrovirus titer was determined and the silencing effects of Pokemon gene in infected Hela cells ex vivo were determined by real time PCR.Results These 4 complementary DNA fragments were successfully cloned into the pSihncer5.1-H1 retrovirus vector separately.The expression level of Pokemon gene was lower in Hela cells that stably transfected with these recombinant plasmids,as compared with normal group.The MTT assay showed that the proliferations of these cells were suppressed.The most effective plasmid was determined and chosen to prepare the recombinant retrovirus.The retrovirus titer was up to 105 cfu/mL,while the expression of Pokemon gene was decreased to about 43% in Hela cells which were infected by the retrovirus.Conclusion The recombinant retrovirus is prepared successfully,which can suppress the proliferation of Hela cell effectively ex vivo.The experimental data provides a powerful proof for cancer therapy strategy of interfering proto-oncogene.