Construction and identification of shRNA lentiviral vector targeting human interleukin-8 gene

Objective:This study aims to target human interleukin-8 gene by constructing shRNA lentiviral vectors and detect its silencing effect in MDA-MB-231 cells.Methods:Two specific siRNA sequences targeting human IL-8 gene were designed and recombined with pMagic7.1lentiviral vector plasmids.The recombined plasmids were identified by PCR and then sequenced.The lentivirus particles were packaged using 293T cells,the titer of which were detected by quantitative DNA technique.Then MDA-MB-231 cells were transfected with the optimized MOI.The silencing effect on IL-8 was detected by real-time PCR.Results:The sequencing of shRNA plasmids which been identified positive by PCR were correctly constructed.The titer of the packaged virus was 263 1.93×109 IU/mL and 1.89×109 IU/mL separately.After infecting the MDA-MB-231 cells with MOI=40,the silencing efficiency were 80% and 90% respectively.Conclusion:The recombinant lentiviral shRNA has been constructed successfully,with a highly silence efficiency,which ensure the reliability for the further study on cell functional assay.