Immortalization of human fibroblasts using tsA mutant of SV40 and pSV3neo plasmid]

Clones of immortalized human fibroblasts with an extended life span in culture and a capability of subloning were obtained after the infection with a temperature sensitive mutant (tsA 239) of SV40 virus and pSV3neo plasmid. As compared with the parental cells, the obtained clones exhibited increased plating efficiency, decreased doubling time, and serum dependence. We did not obtained the colony formation during cultivation of immortalized cells in semiliquid agar. This means that our cells were not completely malignant. The PCR (polymerase chain reaction)-analysis has revealed the presence of viral DNA at early passages (25th passage) after the infection by tsA SV40, and its absence after a prolonged cultivation (46th passage). PCR-analysis of the clones obtained after pSV3neo transfection has revealed the presence of gene A sequences either at early (9-15), or later (62) passages. The expression of the gene A product in cells of these clones was revealed only early passages (11 and 35). Possible mechanisms of immortal phenotype origin in human diploid cells after the action of ts-mutant and other constructions of SV40 are discussed.