[Construction and screening of plasmid expression vectors encoding the short hairpin RNA targeting hVEGF(165) gene].
OBJECTIVE
To construct a plasmid expression vector coding for the short hairpin RNA (shRNA) targeting hVEGF(165) mRNA.


METHODS
Three plasmid expression vectors coding for shRNA targeting exons 5 and 7 of hVEGF(165) gene sequence and a control vector containing random DNA fragment were constructed. The recombinant plasmids were identified by PCR, and then transfected separately into BHK cells expressing hVEGF(165) gene via Viofect(TM) reagent. The hVEGF(165) gene silencing effect was detected by quantitative RT-PCR and Western blotting.


RESULTS
he expected band was amplified from the plasmids coding for shRNA by PCR. Transfection of BHK cells expressing hVEGF(165) gene with the shRNA plasmids resulted in a inhibition of hVEGF(165) mRNA and protein expressions by 77% and 70%, respectively.


CONCLUSIONS
The plasmid expression vectors coding for shRNA targeting hVEGF(165) mRNA have been constructed successfully, of which pDC316- EGFP-U6-shRNA(2) most effectively silences hVEGF(165) gene in BHK cells.
Wang
Wei-Dong
WD
Department of Neurology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. wj120019@msn.com
Jiang
Li-Xin
LX
Xu
Jiang-Ping
JP
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / 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