Lentiviral shRNA Plasmid Targeting Murine TRAF6 and Its Activity of Specific Genetic Interference

Objective:The aim of this study was to establish a lentiviral shRNA plasmid targeting murine TNF receptor-associated factor 6(TRAF6).Method:Specific oligonucleotides for silencing murine TRAF6 were designed and synthesised,and inserted into pENTR/U6 vector following gene sequence analysis.Then,these specific oligonucleotides were cloned into a lentiviral vector CS-RfA-EG based on Gateway LR Clonase Enzyme System.These plasmids were examined with restriction enzyme KpnⅠ.Thereafter,this plasmid was transfected into MEF13 cells under lentivirus infection.The transfection efficiency was determined by flow cytometry.Also,Western blotting was performed to detect the expression of endogenous TRAF6 and phospho-IκBα(p-IκBα)in TRAF6 gene specific shRNA expressing MEF13 cells.The lentiviral shRNA plasmid targeting LacZ was used as control.Result:The constructed lentiviral shRNA plasmid coincided with that of design.The transfection efficiency was over 95%.Endogenous expression of TRAF6 and p-IκBα were completely blocked in murine TRAF6 gene specific shRNA expressing MEF13 cells as compared with that of control.Conclusion:Our results demonstrated that the murine TRAF6 targeted lentiviral shRNA plasmid was successfully constructed.