Development of a mouse cell line containing the phi X174 am3 allele as a target for detecting mutation

Transgenic mice containing multiple copies of the Phi X174 am3 allele are b eing developed as a model for detecting tissue-specific in vivo mutation. I n order to derive an analogous system for measuring am3 mutation in vitro, cells were cultured from 15-day-old C57B1/6J mouse embryos that were homozy gous for the transgene and these cells were transfected with a plasmid expr essing 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-nitro sourea (ENU), assayed for survival by cloning efficiency after overnight cu lture, and assayed for am3 mutations after 5 days of culture. Survival decr eased to 31% at the highest dose of ENU, while mutant frequency increased w ith 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 w ith 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 d eveloping the in vivo am3 mutant assay and for evaluating the sensitivity o f the am3 allele to various classes of mutagens. (C) 1999 Elsevier Science B.V. All rights reserved.