A recombinant plasmid (pMTONCO) containing the coding sequences for rat oncomodulin under the direction of the metallothionein promoter was constructed. pMTONCO was co-transfected with the pSV2-NEO plasmid into primary mouse kidney cells or Rat-1 cells using the calcium phosphate technique and stable transformants were isolated after selection with G418. Transcription from the metallothionein promoter was inducible with heavy metals and produced an oncomodulin-specific mRNA. The presence of oncomodulin protein in stable cell lines was verified by immunoprecipitation with specific antisera. While a plasmid encoding the polyomavirus T-antigens was able to prolong the life-span of primary mouse kidney cells in culture, no equivalent activity was noted when the pMTONCO plasmid was used to transfect primary cells. When expressed in Rat-1 cells, oncomodulin did not affect the growth properties of these cells, nor did it predispose cells to higher frequencies of oncogenic transformation to a viral oncogene. We conclude that oncomodulin is neither an immortalizing nor transforming agent in vitro.

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