Rapid regeneration of virus from cells infected with a retroviral vector

Recombinant retroviral vectors usually encode the genes of interest in place of the viral structural genes, which must be provided in trans. These viruses are therefore defective for replication: infected cells cannot produce progeny virus. However, in some cases it may be desirable to generate virus from an infected cell clone displaying a phenotype of interest. We describe a rapid method for producing virus, which involves fusing the infected cells to fresh packaging cells. Stable producer lines are generated after fusion by co-selecting for the Ecogpt+ marker in the packaging cells and the G418 resistance (neor) marker in the infected cells. We have used this method to develop cell lines that produce retroviruses encoding a Leu 61-activated c-Ha-ras oncogene as well as a neor gene. The viruses confer oncogenic transformation on 95%-100% of infected target cells as assayed by altered morphology, focus formation and soft agar growth.