Requirement for cell dispersion prior to selection of induced azaguanine-resistant colonies of Chinese hamster cells.
With V79 Chinese hamster cell cultures treated with a mutagen, the maximum frequency of colonies resistant to 8-azaguanine (AZG) was attained when the cells were dispersed after a suitable expression time before adding the selection medium. V79-4 cells were exposed to 500 muM MMS, 7 muM AFAA, or 10 muM MNNG and allowed to multiply before being reseeded at 4 times 10-4 cells/60 mm dish and selected with 10 mu-g/ml AZG. Maximum frequencies of 4 times 10-5, 4 times 10-4, and 2.4 times 10-3 were obtained about 100, 130, and 200 hrs after exposure to MMS, AFAA, and MNNG, respectively. The maximum frequencies following MMS or MNNG treatments were about 10-fold greater than those obtained when induction and selection of AZG-resistant colonies were performed in the same culture dish. The reseeding of treated cells eliminated the possibility of metabolic cooperation within mosaic colonies of wild-type and mutant cells and achieved expression of the induced changes before intercolony crossfeeding reduced the frequency of resistant colonies. -  AZG-resistant colonies were selected in medium containing dialyzed fetal bovine serum, and the selection medium replacement were necessary for consistent achievement of background frequencies of resistant colonies near 10-6. Reconstruction experiments with AZG-resistant V79 lines showed that the efficiency of recovery of resistant cells in the selection medium was constant over a range of 0-20 colonies observed/dish. A mixed population of V79 and AZG-resistant cells was also correctly analyzed by the procedure used in mutagenesis studies.
D015073:2-Acetylaminofluorene / Q000494:pharmacology; D000818:Animals; D001375:Azaguanine / Q000494:pharmacology*; D002460:Cell Line; D002478:Cells, Cultured / Q000187:drug effects* / Q000201:enzymology; D006224:Cricetinae; D003470:Culture Media; D003584:Cytological Techniques; D004351:Drug Resistance; D007041:Hypoxanthine Phosphoribosyltransferase / Q000378:metabolism; D066298:In Vitro Techniques; D008168:Lung; D008297:Male; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D009153:Mutagens / Q000494:pharmacology; D009154:Mutation; D011336:Probability; D012641:Selection, Genetic
D003470:Culture Media; D009153:Mutagens; D008769:Methylnitronitrosoguanidine; D015073:2-Acetylaminofluorene; D008741:Methyl Methanesulfonate; D007041:Hypoxanthine Phosphoribosyltransferase; D001375:Azaguanine