Immortalization of primary mouse embryo fibroblasts with SV40 virions, viral DNA, and a subgenomic DNA fragment in a quantitative assay.

In order to investigate the capacity of the N-terminal half of large T antigen to convert primary cells to continuous cell lines conditions were standardized for transforming primary mouse embryo fibroblasts of C57Bl/6 origin (B6/MEF) with DNA. Using these conditions the transforming capability of SV40 virions, viral DNA, and a plasmid, pSV3T3-20-GV (C.E. Clayton, D. Murphey, M. Lovett, and P.W.J. Rigby, Nature (London), 299, 59-61, 1982) containing the 5' half of the SV40 early region, was determined in a quantitative immortalization assay. The plasmid pSV3T3-20-GV transformed B6/MEF at only 1/16 the frequency of a plasmid containing the entire early region. These results suggest that the 3' half of large T antigen which cannot be produced by this plasmid strongly influences the frequency of immortalization. It is not known whether this influence reflects the presence of a transformation domain in the carboxy terminus of large T antigen or, alternatively, results from an altered conformation or stability of the truncated polypeptide.