This project mainly involves the development of techniques for the derivation of continuous cell lines, for use in neural transplantation as an alternative to primary cells and tissues, as well as for studies of neural cell biology. Current efforts include development of mutant truncated forms of SV40 large T antigen, methods for direct cellular delivery of proteins which modify the cell cycle, and sequential delivery of genes to cell lines. A mutant form of SV40 large T antigen, which lacks p53 and DNA binding activity, has been cloned to examine those properties of SV40 large T antigen which are required for immortalizing CNS neurons. This mutant oncogene, called T155, is capable of overcoming cell cycle arrest in T64-7b cells and immortalizing primary mecencephalic neurons when transduced by lipofection. T155 appears to interfere with the expression of differentiated phenotypes to a much smaller degree than wild-type SV40 large T antigen. Primary mesencephalic cell cultures immortalized with T155 express differentiated neuronal markers (e.g., neurofilament) and glial markers (e.g. GFAP), whereas cells immortalized with wild-type SV40 large T rarely express markers characteristic of mature neurons or glia. When T155-immortalized cells are maintained in long-term cultures, they develop variable differentiated morphologies suggestive of mature neurons and glia. Because of the small size of this oncogene, it may be possible to deliver it to cells by forming fusion proteins with other carrier polypeptides. 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. From these experiments we hope to improve the methods for producing immortalized cell lines, especially regarding immortalization techniques for use in neurons.
