A Rapid and Precise Mutation-Activated Fluorescence Reporter for Analyzing Acute Mutagenesis Frequency

Michael D. Birnbaum, Leah Nemzow, Akhilesh Kumar, Feng Gong, Fangliang Zhang

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

Mutagenesis reporters are critical for quantifying genome stability. However, current methods rely on cell survival/death to report mutation, which takes weeks and prevents evaluation of acute or time-dependent changes. Existing methods also have other limitations, such as cell type restrictions. Using our discovery that mCherryFP fluorescence depends on residue Trp98, we replaced this codon with a stop codon to generate a mutation biosensor (termed CherryOFF), with a green fluorescence protein (GFP) as an internal control. We found that the red fluorescence of this biosensor is activated by a specific A/T-G/C nucleotide transition. Compared with the established hypoxanthine phosphoribosyl transferase assay, our reporter has similar or better ability to detect changes of mutation frequency induced by physical/chemical mutagens or manipulation of mutation-related genes. Furthermore, CherryOFF-GFP can report mutagenesis independently of cell-death events, can be adapted to many cell types, and can generate readouts within 1 day for the measurement of acute or time-dependent events. The mutation-activated CherryOFF-GFP reporter quickly and accurately reflects point mutation frequency via flow cytometry readout. This reporter can easily be adapted for different point mutations and indels.

Original languageEnglish (US)
Pages (from-to)1038-1049.e5
JournalCell Chemical Biology
Volume25
Issue number8
DOIs
StatePublished - Aug 16 2018

Keywords

  • GFP
  • assay
  • fluorescence
  • mCherryFP
  • mutagenesis
  • mutation frequency
  • reporter
  • time-dependent dynamic

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmacology
  • Drug Discovery
  • Clinical Biochemistry

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