| Abstract: | The persistence of individual hypoxanthine phosphoribosyltransferase (HPRT)-deficient cells in small populations of mutagenized CHO was examined. Most of these variants were unstable with progressive cultivation in non-selective medium (α) before exposure to the selective agent, thioguanine (TG), but after selection virtually all resistant colonies were stable. The role of cell density in this effect was assessed by shifting sister cultures of low-density populations to high and then back to low density in α-medium and measuring cloning efficiency (CE) in TG after each shift. The high density cells almost always had a lower CE in TG than their low density siblings, indicating a relative loss of TG resistance. When they were passed again at low density, the higher CE of the sister culture was usually not reacquired. These variants therefore appeared to be sensitive to a density-dependent reversal of phenotype. This interpretation was verified by growing sister cultures of biochemically marked, mutagenized CHO cells in TG for 3 days. The resistant colonies were then grown in α-medium and challenged by co-incubating colonies of one dish with wild-type (WT) unmarked cells immediately and those of the sister dish with WT after various periods in α-medium. Most TG-resistant colonies underwent some degree of stable reversal to the HPRT+ phenotype when challenged immediately, but their sister colonies, tested at later times, became insensitive to this effect over periods ranging up to 30 days or more after mutagenesis. |
