Two approaches have been used by this laboratory for the isolation of new oncogenes: (1) transduction with retrovirus and (2) transformation of cells by retrovirus insertion followed by molecular cloning of flanking DNA. The first approach has yielded the v-raf oncogene carrying virus 36llMSV and several other isolates which will be molecularly analyzed in the future. For the second approach an in vitro system was established for the transformation of rat epithelial cells (RC-E) by C3H MuLV in conjunction with 12-0-tetradecanoylphorbol-13-acetate (TPA). Transformed cell clones obtained from soft agar generally were virus non-producers. The purpose of these experiments was to sequence-label TPA-promotable cellular tumor genes, and thus, make possible their isolation by molecular cloning. RC-E cells that had been transformed with MuLV and TPA are being examined for expression of novel RNA transcripts initiating in the viral LTR. Those non-producers which contain novel virus-cell hybrid transcripts are being used for the molecular cloning of LTR-linked cellular DNA. As an example for the principle effectiveness of such a strategy, we have demonstrated for NIH 3T3 cells transformed by transfection with MuLV LTR DNA that the proto-oncogene, c-raf-l, was activated as an oncogene by a promoter insertion mechanism.
