Embryonal carcinoma cells have proven to be of particular value in studies of both oncogenesis and mammalian development as well as in evaluating the relationship between these two phenomena. We have infected embryonal carcinoma cells with a retrovirus in an effort to create insertion mutations which result in cells defective in either their differentiative or oncogenic potentials. One such cell line, originally identified by its unique morphological phenotype, is abnormal in respect to both parameters. These cells do not differentiate along typical embryonal carcinoma stem cell lineages possibly having lost their ability to elaborate endodermal derivatives. Most significantly, they have also lost their ability to form tumors in syngeneic mice. Southern blot analysis indicates that this variant cell line has a single viral insert and the original cell was probably hemizygous for the insertion site. In addition we have recently isolated a revertant cell line which appears to have arisen through spontaneous excision of the retrovirus. Therefore, if the variant is indeed a consequence of an insertional mutation, a single gene may regulate both the tumorigenic and differentiative capacities of the cell. It is the purpose of this proposal to explore this possibility by identifying and cloning this particular gene. To this end the genomic sequence flanking the provirus will be isolated. Within the sequence, a transcript will be identified that shows a difference between the parental and variant cells. The corresponding cDNA will be isolated and used in a DNA transfection assay to examine the causal relationship between insertion and phenotypic change. Alternatively, genes differentially expressed between the two cell lines will be isolated by the method of differential-cDNA cloning and similarly analyzed. The differentiative potentials of the parental and variant cell lines will be examined and compared both in vitro utilizing molecular, biochemical and immunological marking techniques and in vivo through the production of blastocyst injection chimeras. Eventually, biochemical alterations associated with the variant phenotypes will be characterized. Analyses of the results obtained should certainly enhance our knowledge of the cellular and molecular elements controlling differentiation and oncogenesis as well as the possible connection between these two phenomena.
