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

Abstract

A HeLa cell line stably expressing the enhanced green fluorescence protein (EGFP) gene, interrupted by the HBB IVS2-654 intron, was studied without treatment and after treatment with a single standard dose of 15 microM of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This assay was done in order to prove that such a construct can revert by a variety of mechanisms and that it produces a visible phenotype, i.e., green fluorescence. The system permits visual detection of living mutant cells among a background of non-mutant cells and does not require a selective medium. The results show that the construct reverts by large deletions (-62, -100, and -162 bp), small insertions (+4 bp), small rearrangements (19 bp duplication), base substitutions at purines (G652, G653, A655, G579), and a pyrimidine (T654) between nucleotide positions 579 and 837. Splice-site mutations were recovered, and some of the mechanisms underlying these mutations are discussed. Because of the ease of detection of revertant cells under fluorescent light and the wide variety of mutations that can be recovered, further development of this system could make it a useful new mammalian cell mutagenicity assay.