A safe packaging line for gene transfer: separating viral genes on two different plasmids

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Abstract
A retrovirus packaging cell line was constructed by using portions of the Moloney murine leukemia virus in which the gag, pol, and env genes of the helper virus were separated onto two different plasmids and in which the .psi. packaging signal and 3'' long terminal repeat were removed. The plasmid containing the gag and pol genes and the plasmid containing the env gene were cotransfected into NIH 3T3 cells. Clones that produced high levels of reverse transcriptase and env protein were tested for their ability to package the replication-defective retrovirus vectors .DELTA.neo and N2. One of the gag-pol and env clones (GP+E-86)was able to transfer G418 resistance to recipient cells at a titer of as high as 1.7 .times. 105 when it was used to package .DELTA.neo and as high as 4 .times. 106 when it was used to package N2. Supernatants of clones transfected with the intact parent gag-pol-env plasmid 3P0 had comparable titers (as high as 6.5 .times. 104 with .DELTA.neo; as high as 1.7 .times. 105 with N2). Tests for recombination events that might result in intact retrovirus showed no evidence for the generation of replication-competent virus. These results suggest that gag-pol, and env, when present on different plasmids, may provide an efficient and safe packaging line for use in retroviral gene transfer.