Generation of high titer pseudotyped GFP-retroviruses by co-transfaction of VSV-G gene

Objective To obtain high liter pseudotyped retroviruses containing GFP gene coatedwith vesicular stomatitis virus G glycoprotein, which could be effectively concentrated by ultracen trifugation. Methods PT67 packaging cells were transfected with the plasmid pGFP-RV followed by selection with G418. The viral-containing super-natants of PT67 cells and the plasmid containing VSV-G gene were co-transfected into GP2-293 packaging cells that were transiently selected by G418. To determine virus liter, NIH 3T3 cells were infected with the pseudotyped retro-viruses, and colonies were scored after selection. By ultracentrifugation, the pseudotyped retroviruses were further concentrated to increase the efficiency of gene transfer. Results Expression of GFP in PT67 cells transfected with pGFP-RV alone declined during long term culture. GP2-293 cells could produce high liter pseudotyped retroviruses containing GFP gene coated with vesicular stomatitis virus G glycoprotein [ (7.5±1.4)×105cfu/mL) ]. The pseudotyped relroviruses concentrated by ultracentrifugation mediated stable and efficient gene transfer. Conclusion Generation of recombinant retroviruses containing GFP gene pseudotyped with vesicular stomatitis virus G glycoprotein may have important value in tissue engineering as a molecular reporter with high efficiency.