Clonal nature of spontaneously immortalized 3T3 cells

Mouse embryo fibroblasts (MEFs), when plated at appropriate densities, proliferate vigorously for several passages, and then the growth rate of the culture slows considerably. If the cells are plated at a high enough density and continuously passed, the cultures will eventually overcome this "crisis" period and resume rapid growth. Here, we have addressed the question of what the changes are that cells undergo in overcoming the growth restraints of crisis. Primary MEF cells were infected with a retrovirus which confers G418 resistance and selected in G418. The resultant pre-crisis population comprised cells which each contained a retrovirus integrated at a unique genomic location. These cells were then passed according to the 3T3 protocol until immortal, rapidly growing cells emerged. The integration pattern of the retrovirus in the immortal population was examined. In two independent experiments, the immortal population of cells grown in the presence of G418 comprised two independent clones of cells, with additional clones undetectable at the level of detection of the assays used. The integration pattern was also examined in parallel infected cultures grown in the absence of selection. In one experiment the unselected immortal population contained the same labeled clone that appeared in the sister infected culture, indicating that an immortal precursor was present in the precrisis population. These results are consistent with the idea that a mutation is responsible for the immortal phenotype.