[Construction and identification of lentiviral vector mediated GATA-3 gene RNA interference in mouse]

Objective:The aim of this study was to construct and identify a lentiviral vector for RNA interference of mouse GATA-3.Method:The coding region of the GATA-3 gene sequences were analyzed for a single-stranded oligo design and synthesis.PCR was performed for splicing reaction of synthetic oligo,which was synthesized into the lentiviral vector and transformed into the competent cell DH5α.An expression vector shRNA-GATA-3 was constructed after sequencing and identifying the gene sequence of the recombinant clones.Accoding to the target gene sequence ,four pairs are as following: siRNA-685 group(LV3-GATA-3-Mus-685),siRNA-1152 group (LV3-GATA-3-Mus-1152),siRNA-1615 group(LV3-GATA-3-Mus-1615) and control group ,which were designed and syntheized and builded into the lentiviral carrier.The shRNA vectors were cotransfected with expression vectors into the 293T cell line,and the optimal interference sequence was screened by QPCR. The 293T cells were cotranfected with the constructed lentiviral interference vectors and packing plasmids,the viruses were packed,the virus stock was collected and concentrated by ultracentrifugation,and the titer of the viruses was determined. Result:Gene sequence proved that the recombinant clone gene sequence was correct,and the lentiviral vector was successfully constructed. The relative expression of GATA-3 mRNA in siRNA-1615 group(0.004) was significantly reduced after RNA interference comparing with that in the control group(0.022),siRNA-1152 group(0.009) and siRNA-685 group(0.009). The screened LV3-GATA-3-Mus-1615 ,as the most effective interference sequence,was constructed into the lentiviral vector.The titer of the concentrated virus of LV3-GATA-3-Mus-1615 was 5×108 TU/ml in the viral supernate of 293T cells collected at 48 hours after co-transfection. Conclusion:Alentiviral RNAi expression vector targeting the GATA-3 gene (LV3-GATA-3-Mus-1615) was successfully constrcted and identified.