Construction of VEGF gene-targeted small interfering RNA mediated by CMV promoter and its silencing effect / 浙江大学学报·医学版

ABSTRACT

<p><b>OBJECTIVE</b>To construct VEGF gene-targeted small interfering RNA (siRNA) and its expression vector driven by CMV promoter and to investigate its interference effect.</p><p><b>METHODS</b>The VEGF gene-targeted hairpin siRNA was designed, two complementary oligonucleotide strands were synthesized. After annealing, two-strand oligonucleotide was inserted into pDC311-SV40-RC vector, which was then identified by PCR and sequenced. Then human U-2 OS cell line was transfected with the vector using lipofectamine method. Finally, ELISA was performed to evaluate the expression of VEGF protein.</p><p><b>RESULTS</b>PCR-identification of positive clone and sequencing confirmed the vector containing the target siRNA. ELISA showed that compared with the control group, the expression levels of VEGF protein in transfected U-2 OS cells were decreased significantly (P<0.05).</p><p><b>CONCLUSION</b>VEGF gene-targeted siRNA and its vector mediated by CMV promoter were successfully constructed, which can reduce the VEGF protein expression after transfecting.</p>