Validation of conditions for efficient detection of HPRT and APRT mutations in suspension-cultured chinese hamster ovary cells

Abstract

Conditions for reliable and efficient assay of mutations affecting the activity of HPRT (hypoxanthine phosphoribosyltransferase EC 2.4.2.8) and APRT (adenine phosphoribosyltransferase EC 2.4.2.7) have been determined for a strain of CHO (Chinese hamster ovary) cells that has been adapted for rapid growth both in suspension culture and in monolayer. To facilitate measurement of mutation at the aprt locus, clones were derived that are presumptively heterozygous at that locus. At a limiting concentration of 8 μg/ml of azaadenine, 14 16 of the resistant clones picked and tested had ∼ 1 2 of the APRT activity of the wild-type cells. One such clone, strain AA8, was chosen for further studies and found to be readily mutable to resistance to 80 μg/ml azaadenine. Most of the highly resistant colonies isolated ( 21 24 ) had very low in vitro APRT activity. The optimal conditions for detection of TG r and AA r mutations were determined for two critical parameters, expression time and cell density. Cultures treated with mutagen either in monolayer or in suspension were allowed to express mutations in suspension. The expression of mutations induced by UV light, EMS, and ICR-191 was complete by 3 days for AA r and by 4–5 days for TG r. The time required to reach a maximal frequency of mutants was essentially independent of the type of mutagen and the level of survival after treatment. Induced mutation frequencies for both loci were notably stable during the time intervals examined. With respect to cell-density conditions, both markers were detected at frequencies that were independent of the cell inocula over the range of 1 x 10 5 to 1 x 10 6 cells per 100-mm petri dish (i.e. 1.6 x 10 3 to 1.6 x 10 4 cells/cm 2) containing 20 ml of medium. These results were obtained with both mutagenized populations and with reconstructed mixtures obtained by adding drug-resistant cells to varying numbers of wild-type cells. The rapid expression of mutations for both markers, particularly AA r, combined with the advantage that large inocula can be plated for selection of mutants, make this CHO strain an attractive system for the simultaneous measurement of mutations at the autosomal aprt and X-linked hprt loci.