High-efficiency gene transfer into mammalian cells: generation of helper-free recombinant retrovirus with broad mammalian host range.

Abstract

A chimeric retrovirus genome containing ecotropic gag-pol sequences was constructed from Moloney murine leukemia virus and envelope sequences derived from the amphotropic virus 4070A. This reconstructed genome, termed pMAV-.psi.-, lacks the .psi. site required for encapsidation of the viral genome. NIH 3T3 cells transfected with pMAV-.psi.-, called .psi.-AM lines, are capable of producing high titer stocks of helper-free recombinant retrovirus with amphotropic host range after transfection with recombinant retroviral vectors carrying the neomycin phosphotransferase gene. Most transfected .psi.-AM cells remain helper-free, even after months in culture. .psi.-AM virus stocks infect nearly all human and murine cell lines tested thus far, as assayed by resistance to the neomycin analog G418. Southern and RNA blot analyses of .psi.-AM-infected human cells show that recombinant murine retroviruses integrate randomly into genomic DNA as normal proviruses and express high levels of the subgenomic and genomic viral messages in the expected stoichiometry of 1:1.