Abstract:

To construct a recombinant lentiviral expression vector carrying small interference RNA (siRNA) with the mouse endothelial vascular growth factor a (VEGFa) gene serving as the target. Methods  ① Three target sequences of mouse VEGFa gene and an irrelevant sequence were designed. Four couples of complementary oligonucleotides with hairpin loop of siRNA were synthesized. After annealing, the DNA fragments were ligated into the plasmid pRNATU6.2/Lenti. ② VEGFa cDNA was cloned from NIH/3T3 strain and subcloned into the plasmid pKCDNA-EF1-Puro. ③ The pCDNA-VEGF plasmid and siRNA lentiviral plasmids were co-transfected into the NIH/3T3 cells and assigned into six groups, the effects of RNAi on VEGFa expression were determined 48h later; ④ The lentiviruses were produced by 293T cells following cotransfection of the pRNAT-siRNA3 plasmid or the pRNAT-negative plasmid with three package plasmid compounds. After 48h, the supernatant was collected and the recombinant lentiviruses titers were determined. Results ① Three lentiviral plasmids of siRNA specific to the VEGFa gene and one negative plasmid were constructed. ② The plasmid pCDNA-VEGF was constructed. ③ In the pRNAT-siRNA3 plasmid cotransfection group, expression of VEGFa mRNA and protein was decreased by approximately 80.0% and 66.3%, respectively.④ The lentivirus titer reached 1×108ifu/ml. Conclusion  The recombinant lentiviruses,which express siRNA hairpin aiming at mouse VEGFa gene, have been constructed.

Key words: Vascular endothelial growth factor; RNA interference; Small hairpin RNAs; Lentivirus