Abstract
We have constructed infectious retroviral vectors, derived from Moloney murine leukemia virus, that efficiently transduce the polyoma virus tumor (T) antigens individually. The parental vector we have chosen [pZIP-NeoSV(X)1] expresses a dominant selectable marker for neomycin resistance and is a shuttle vector capable of propagation in both eukaryotic and prokaryotic cells, thus facilitating its use in structure-function studies. To address the relationship between polyoma T-antigen tumorigenesis and the effects of individual T antigens on growth control and differentiation, we used these vectors to introduce and stably express large, middle-sized, or small T antigens into mouse fibroblasts and preadipocytes. All cDNAs introduced into the vector are expressed stably even in the absence of selective pressure. The stable expression of small T antigen is noted particularly because cell lines expressing small T antigen have not been readily available prior to the use of retroviral vectors. Small T antigen-induced increase in saturation density of NIH 3T3 cells can be scored on the basis of the morphology of drug-resistant colonies. Middle-sized T antigen eliminates the growth requirement of NIH 3T3 cells for epidermal growth factor in a defined medium and permits growth in platelet-poor plasma, indicating elimination of the platelet-derived growth factor requirement as well. Large T antigen suppresses mouse preadipocyte (3T3-F442A) differentiation. These vectors and these functional assays of T-antigen activity permit genetic analysis of the relationship between tumorigenesis by T antigens and the alteration of cellular growth and differentiation.
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