Clones of Chinese hamster cells cultivated in vitro not permanently resistant to azaguanine

The frequency of clones not permanently resistant to azaguanine (AG) was measured in Chinese hamster ovary cells (CHO) grown in vitro by plating them in 7.5 μg/ml AG and isolating a number of clones in the course of 5 experiments. Such isolated clones were propagated to a point at which their resistance to both AG and the reverse selective medium, HAT, could be determined. Out of a total of 13 clones isolated, 4 of these could not be distinguished from the parent CHO line, either on the basis of their growth in a gradient of AG concentrations or the reverse selective HAT medium or on the basis of their mutation frequency to resistance to 30 μg/ml AG. All four of the apparent phenocopies were isolated from plates in which although lower numbers of cells were seeded, a higher frequency of clones able to grow in AG was yielded. This suggests that the higher “mutation” frequencies obtained at lower cell densities are due to the appearance of phenocopies which occur only under these conditions. It is concluded that under low plating density conditions, the lower levels of AG (7.5 μg/ml) are not satisfactory for mutagenesis and mutation rate studies.