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 and immortalizing primary mecencephalic neurons. 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. A further series of variants of the T155 oncogene have been developed in order to obtain the minimal oncogene required for immortalization of neuronal precursor cells. We also have developed methods for inducing immortalized striatal neurons to produce high levels of GABA, and these cells are effective in transplantation studies in animals. These studies may lead to the production of human cell lines that could be used for therapeutic purposes and as in vitro models for studying effects of drugs of abuse. - immortalized cell lines, SV40 large T antigen, T155, p53, in vitro, neuronal, glial
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