[Construction of VEGF gene-targeted small interfering RNA mediated by CMV promoter and its silencing effect].
To construct VEGF gene-targeted small interfering RNA (siRNA) and its expression vector driven by CMV promoter and to investigate its interference effect.



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.



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).



VEGF gene-targeted siRNA and its vector mediated by CMV promoter were successfully constructed, which can reduce the VEGF protein expression after transfecting.
Liu
Xiao-yan
XY
The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
Fang
Hong
H
Chen
Hong-chao
HC
Jiang
Xiao-ling
XL
D000260:Adenoviruses, Human / Q000235:genetics; D001483:Base Sequence; D001859:Bone Neoplasms / Q000473:pathology; D045744:Cell Line, Tumor; D020868:Gene Silencing; D006801:Humans; D008969:Molecular Sequence Data; D012516:Osteosarcoma / Q000473:pathology; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
D012333:RNA, Messenger; D034741:RNA, Small Interfering; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A