Efforts are currently being devoted to the derivation of continuous cell lines, which can be maintained in tissue culture, for use in neural transplantation as an alternative to primary cells and tissues. We have used SV40 large T antigen to immortalize cells from primary CNS cultures, using methods to select for cells with a neuronal lineage. GABA- producing striatal cell lines, and mesencephalic cell lines which respond to basic FGF, have been obtained. In most cases, however, the viral oncogenes that are used to immortalize cells interfere with cell cycle control, so that immortalized cells do not become terminally differentiated. Ultimately, it will be necessary to improve the methods for immortalizing neurons, so that cell lines can both be propagated in culture, and also induced to exit from the cell cycle to allow terminal differentiation. We have studied cells immortalized with a temperature- sensitive form of SV40 large T antigen, and found that such cells continue to divide even at the non-permissive temperature, unless provided with additional stimuli (e.g., confluence) to inhibit cell growth. A mutant form of SV40 large T antigen, which lacks p53 binding activity, has been cloned to examine those properties of SV40 large T antigen which are required for immortalizing CNS neurons. Alternative means of intervening in cell cycle control, using direct delivery of protein factors and antisense approaches, are being studied alone and in combination with SV40 large T mutants for the complementary purposes of developing an improved understanding of the steps required for cell immortalization, and improving the methods for producing immortalized cell lines.
