Development of a mouse cell line containing the PhiX174 am3 allele as a target for detecting mutation

Transgenic mice containing multiple copies of the PhiX174 am3 allele are being developed as a model for detecting tissue-specific in vivo mutation. In order to derive an analogous system for measuring am3 mutation in vitro, cells were cultured from 15-day-old C57Bl/6J mouse embryos that were homozygous for the transgene and these cells were transfected with a plasmid expressing the SV40 large T-antigen. Two G418-resistant colonies were isolated from this culture and expanded to continuously proliferating cell lines (PX-1 and PX-2). Line PX-2 was treated with up to 1.0 mg/ml of N-ethyl-N-nitrosourea (ENU), assayed for survival by cloning efficiency after overnight culture, and assayed for am3 mutations after 5 days of culture. Survival decreased to 31% at the highest dose of ENU, while mutant frequency increased with dose from approximately 2 x 10(-7) in the untreated cells to 13 x 10(-7) in cultures treated with 0.6 mg/ml of ENU. PX-2 cells also were treated with 0 and 0.6 mg/ml of ENU and mutant frequency assays were performed after 5, 24, 48 and 72 h of growth. The mutant frequency in the treated culture increased to 20 x 10(-7) at 48 h and remained approximately the same at 72 h. These results indicate that PX-2 cells should be a useful resource for developing the in vivo am3 mutant assay and for evaluating the sensitivity of the am3 allele to various classes of mutagens.