Construction of lentivirus-mediated RNAi vector targeting vasodilator-stimulated phosphoprotein
WANG Jing1 WEI Lei2 QIU Kun1
1.Department of Pathology, Wuhan No.1 Hospital, Hubei Province, Wuhan 430022, China;
2.Department of Pathology and Pathophysiology, School of Basic Medical Sciences, Wuhan University, Hubei Province, Wuhan 430071, China
Abstract：Objective To construct the lentiviral RNA interference expression vector targeting vasodilator-stimulated phosphoprotein (VASP) gene. Methods The pre-constructed pcDNA6.2-miRVASP was identified by DNA sequencing and the positive clones were screened. The miRVASP fragment was cloned into destination vector pLenti6/V5-DEST by Gateway techology, including BP and LR reaction, RNA interference lentiviral vector pLenti6/V5-miRVASP targeting VASP gene was constructed. Then the 293FT cells were co-transfected with the plasmid lentiviral expression vector pLenti6/V5-miRVASP and lentiviral packaging mix. The virus particles were collected and infected into gastric cancer BGC-823 cells. Results The constructed lentiviral expression vector pLenti6/V5-miRVASP was introduced into E.coli Stbl3 for amplification. DNA sequencing results showed that the constructed vector was exactly as expected. BGC-823 cells were infected with the collected lentiviral stocks carrying the miRVASP expression cassette. The results showed that a large number of cells could express strong green fluorescence, demonstrated that the packaged lentiviral stocks could infect BGC-823 cells. Conclusion The RNA interference lentiviral vector targeting VASP gene is successfully constructed and packaged as the lentiviral stocks to successfully infect BGC-823 cells, which lay the foundation for the further study of the role of VASP in gastric cancer metastasis in vivo.
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2018, Vol. 15 
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