Construction and identification of a recombinant lentivirus harboring shrna targeting rat STAT3 gene

Objective Based on the 4 plasmids expressing shRNA targeting STAT3 successfully constructed earlier,to screen the one with best interference effect,and conduct lentivirus packing and identification.Methods The target cells(rat mesangial cells) were transfected with the 4 STAT3 shRNA plasmids: shRNA1,shRNA2,shRNA3,shRNA4 and the empty vector control(Control1),invalid shRNA control(Control2).mRNA was extracted and expression of STAT3 was measured by RT-PCR after 24 hours of incubation.Lentivirus packing was done on the one with best interference effect using pPACKH1TM packaging plasmids.The titer and infection efficiency of the recombinant lentivirus were determined according to the expression of the reporter gene enhanced green fluorescent protein(EGFP) uner fluorescent microscope.Results RT-PCR results showed that shRNA3 was the most effective in STAT3 interference.A recombinant lentivirus Lenti-shRNA3 was successfully constructed and co-transfected into 293T cells.24h and 48h later,green fluorescence were detected under inverted fluorescence microscope.Then Lenti-shRNA3 was tranfected into HT1080 cells,the supernatant was collected and the titer of the concerned virus was 3.34×108ifu/ml.Conclusion Recombinant lentivirus plasmid of RNA interference targeting rat STAT3 gene was successfully constructed,which could suppress STAT3 expression with high efficiency,and can be used in further study.