[Construction and screening of plasmid expression vectors encoding the short hairpin RNA targeting hVEGF(165) gene]
Wei-Dong Wang1, Li-Xin Jiang, Jiang-Ping Xu
1Department of Neurology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. wj120019@msn.com
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.
Conclusion:
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.
