GROWTH-PROPERTIES OF NEURAL CELL-LINES IMMORTALIZED WITH THE TSA58 ALLELE OF SV40 LARGE T-ANTIGEN

In vitro growth properties of three CNS-derived cell lines were compar ed under a variety of culture conditions, The M213-2O and J30a cell li nes were each derived from embryonic CNS culture with the temperature- sensitive (ts) allele of SV40 large T antigen, tsA58, while the A7 cel l line was immortalized using wild-type SV40 large T antigen, Cells im mortalized with tsA58 SV40 large T proliferate at the permissive tempe rature, 33 degrees C, while growth is expected to be suppressed at the nonpermissive temperature, 39.5 degrees C, Both the M213-20 and J30a cell lines were capable of proliferating at 39.5 degrees C continuousl y for up to 6 mo, All three cell lines showed no appreciable differenc es in growth rates related to temperature over a 7-day period in eithe r serum-containing or defined serum-free media, The percentage of cell s in S-phase of the cell cycle did not decrease or was elevated at 39. 5 degrees C for all three cell lines, After 3 wk at 39.5 degrees C, th e three cell lines also showed positive immunostaining using two monoc lonal antibodies reacting with different epitopes of SV40 large T anti gen, Double strand DNA sequence analyses of a 300 base pair (bp) fragm ent of the large T gene from each cell line, which included the ts loc us, revealed mutations in both the J30a and M213-2O cell lines, The J3 0a cell line ts mutation had reverted to wild type, and two additional loci with bp substitutions with predicted amino acid changes were als o found, While the ts mutation of the M213-20 cells was retained, an a dditional bp substitution with a predicted amino acid change was found , The A7 cell line sequence was identical to the reference wild-type s equence. These findings suggest that (a) nucleic acid sequences in the temperature-sensitive region of the tsA58 allele of SV40 large T are not necessarily stable, and (b) temperature sensitivity of cell lines immortalized with tsA58 is not necessarily retained. (C) 1997 Elsevier Science Inc.