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Published on: June 9, 2009
[Construction of VEGF gene-targeted small interfering RNA mediated by CMV promoter and its silencing effect]
Xiao-yan Liu1, Hong Fang, Hong-chao Chen
1The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
Objective:
To construct VEGF gene-targeted small interfering RNA (siRNA) and its expression vector driven by CMV promoter and to investigate its interference effect.
Methods:
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.
Results:
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).
Conclusion:
VEGF gene-targeted siRNA and its vector mediated by CMV promoter were successfully constructed, which can reduce the VEGF protein expression after transfecting.
