Flow cytometric detection of tandem repeat mutations induced by various chemical classes

Mutation Research
Caroline HealyCraig Parfett

Abstract

To facilitate detection of genotoxicity from environmental mutagen exposure, we generated an in vitro enhanced green fluorescence protein (EGFP) reactivation assay that quickly and effectively detects frameshift mutations in tandem repeat sequences (TRS). Two murine cell lines, C3H10T1/2 and mismatch repair deficient MC2a, were stably transfected with EGFP reporter plasmids in which the EGFP constructs contain TRS that put the EGFP sequence out of frame. These included several 2, 3, 4, 5 and 6 bp repeat sequences, a control non-repetitive sequence and a human gene sequence containing a 4 bp repeat motif. Transfected cultures were exposed to five model mutagens and carcinogens: hydrogen peroxide (H(2)O(2)), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), benzo-a-pyrene-diol-epoxide (BPDE), ethyl nitrosourea (ENU), 9-aminoacridine (9AA) and two controls: acetone and ethanol. Frameshift mutations resulted in green fluorescent revertants, as determined by flow cytometry, and were confirmed, for 9AA treatments, by sequencing. All five treatments with model agents induced statistically significant sequence- and exposure-dependent responses in MC2a cells and a negative response with the two negative control treatments, acetone and ethano...Continue Reading

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Citations

May 24, 2013·Molecular Genetics and Genomics : MGG·Xiaoyan DuZhenwen Chen
Apr 8, 2010·Cell Stress & Chaperones·David MittelmanJohn H Wilson
Jun 4, 2010·Cell Stress & Chaperones·David Mittelman, John H Wilson
May 15, 2007·Mutation Research·Lynnette R Ferguson, William A Denny
Jul 26, 2008·Aging Cell·Jared M Fischer, James R Stringer

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