Construction and effect identification of VEGF-C eukaryotic expression vector for RNA interference

Objective:To construct VEGF-C eukaryotic expression vector for RNA interference. Methods: Recombinant were designed and established by targeting gene VEGF-C and plasmid pGENSIL-1 based on VEGF-C cDNA sequences of genomes, two pairs of oligonucleotides were synthesized according to the Tuschl and inserted into plasmid pGenSil-l to generate siRNA eukaryotic expression vector, DH5α strains were transformed, plasmid were extracted, and recombinant vector were identified by the restriction map and the sequence analysis. The recombinant plasmid(pGensil-VEGF-C1 and pGensil-VEGF-C2 )was transfected into the cultured LOVO cells . At 48h after transfection, the whole cell protein and RNA were extracted, and the protein level was detected by Western blot with mouse -anti-human VEGF-C monoclonal antibody, the mRNA level of VEGF-C was detected by RT-PCR. Results: Recombinant plasmids were completely coincided with the designs by the restriction map and the sequence analysis, pGenSil-VEGF-C expression vector into LOVO cells down -regulated the protein level of VEGF-C at 48h after transfection ,the recombinant eukaryotic expression vector were constructed successfully. Conclusion: siRNA recombinant can be constructed successfully by RNAi technique for inhibition VEGF-C expression.