A TRUNCATED SV40 LARGE T-ANTIGEN LACKING THE P53 BINDING DOMAIN OVERCOMES P53-INDUCED GROWTH ARREST AND IMMORTALIZES PRIMARY MESENCEPHALIC CELLS

As an alternative to primary fetal tissue, immortalized central nervou s system (CNS)-derived cell lines are useful for in vitro CNS model sy stems and for gene manipulation with potential clinical use in neural transplantation. However, obtaining immortalized cells with a desired phenotype is unpredictable, because the molecular mechanisms of growth and differentiation of CNS cells are poorly understood. The SV40 larg e T antigen is commonly used to immortalize mammalian cells, but it in terferes with multiple cell-cycle components, including p53, p300, and retinoblastoma protein, and usually produces cells with undifferentia ted phenotypes. In order to increase the phenotypic repertoire of immo rtalized CNS cells and to address the molecular mechanisms underlying immortalization and differentiation, we constructed an expression vect or containing a truncated SV40 large T gene that encodes only the amin o-terminal 155 amino acids (T155), which lacks the p53-binding domain. Constructs were first transfected into a p53-temperature-sensitive ce ll line, T64-7B. Colonies expressing T155 proliferated at the growth-r estrictive temperature. T155 was then transfected into primary culture s from embryonic day-14 rat mesencephalon. Two clonal cell lines were derived, AF-5 and AC-IO, which co-expressed T155 and mature neuronal a nd astrocytic markers, Thus, the amino-terminal portion of SV40 large T is sufficient to: (1) overcome p53-mediated growth arrest despite th e absence of a p53-binding region, and (2) immortalize primary CNS cel ls expressing mature markers while actively dividing. T155 and T155-tr ansfectants may be useful for further studies of cell-cycle mechanisms and phenotyic expression in CNS cells or for further gene manipulatio n to produce cells with specific properties.