Spontaneous and MNNG-induced reversion of an EGFP construct in HeLa cells:An assay for observing mutations in living cells by fluorescent microscopy

A HeLa cell line stably expressing the enhanced green fluorescence protein (EGFP) gene, interrupted by the HBB IVS2-654 intron, was studied without tr eatment and after treatment with a single standard dose of 15 muM of N-meth yl-N'-nitro-N-nitrosoguanidine (MNNG). This assay was done in order to prov e that such a construct can revert by a variety of mechanisms and that it p roduces a visible phenotype, i.e., green fluorescence. The system permits v isual detection of living mutant cells among a background of non-mutant cel ls and does not require a selective medium. The results show that the const ruct reverts by large deletions (-62, -100, and -162 bp), small insertions (+4 bp), small rearrangements (19 bp duplication), base substitutions at pu rines (G(652), G(653), A(655), G(579)), and a pyrimidine (T-654) between nu cleotide positions 579 and 837. Splice-site mutations were recovered, and s ome of the mechanisms underlying these mutations are discussed. Because of the ease of detection of revertant cells under fluorescent light and the wi de variety of mutations that can be recovered, further development of this system could make it a useful new mammalian cell mutagenicity assay. Hum Mu tat 18:526-534, 2001. (C) 2001 Wiley-Liss, Inc.