Construction and identification of lentiviral vector of RNA interference of Smad4 gene

AIM: To construct a lentiviral vector of RNA interference (RNAi) of Smad4 gene. METHODS: The effective sequence of siRNA targeting Smad4 gene was confirmed in our previous study. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and cloned into the pLVTHM vector, which contained H1 promoter and green fluorescent protein (GFP). The resulting lentiviral vector containing Smad4 shRNA was named LV- shSmad4, and it was confirmed by PCR and sequencing. 293T cells were cotransfected with lentiviral vector LV-shSmad4,pCMV-dR8.74 and pMD2G. All virus stocks were produced by calcium phosphate-mediated transfection. The titer of virus was tested according to the expression level of GFP. RESULTS: PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of Smad4 (LV-shSmad4) producing Smad4 shRNA was constructed successfully. The titer of concentrated virus was 5×10 10 pfu/L. CONCLUSION: The lentivirus RNAi vector of Smad4 was constructed successfully.