This project mainly involves the development of techniques for the derivation of continuous cell lines, for use as models to investigate effects of drugs in vitro as well as for use in neural transplantation as an alternative to primary cells and tissues, as well as for basic 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 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 T64-7bcells 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, and, moreover avoid the problem of interference with the activity of p53 which could promote the survival of cells with DNA damage. Primary mesencephalic cell cultures imortalized 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 on long-term cultures, they develop variable differentiated morphologies suggestive fo 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. We now have a retroviral vector capable of delivering p53, which will be employed to produce other immortal cell types, possibly including human neurons. Alternative means of intervening in cell cycle control, using direct delivery of protein factors and antidense approaches, are being studied alone and in combination with SV40 large T mutants. We also have developed methods for inducing immortalized striatal neurons to produce high levels of GABA, which could result in the production of human cell lines that could be used for theapeutic purposes. From these experiments we hope to improve the methods for producing immortalized cell lines, especially regarding immortalization techniques for use in neurons.
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