Construction and identification of lentivirus of RNA interference targeting VEGF165 gene

Objective To construct a lentiviral vector of RNA interfence(siRNA) of VEGF165 gene,and preparing mature virus.Methods The effective sequence of siRNA targeting VEGF165 was confirmed in our previous study.The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was synthesized and cloned into the PGC SIL-GFP lentivector obtaining the VEGF165 siRNA virus vector named pGCSIL-SiVEGF.293T cells were cotransfected with pGCSIL-SiVEGF and lentivirus packaging plasmids mixtures to prepare the mature virus named LV-SiVEGF.The titer of virus was tested according to the expression level of GFP.The human umbilical vein endothelial cell(HUVEC) was transfected by the LV-SiVEGF.We detected expression levels of VEGF mRNA of HUVEC by RealTime-PCR.Results PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of VEGF was constructed successfully.The titer of concentrated virus was 2×109 ifu/μl.VEGFmRNA expression levels of the experimental HUVEC were significantly lower than the control group(73.3%).Conclusions VEGFsiRNA lentiviral was successfully constructed.