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 journalArticle

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)
JournalCell Chemical Biology
DOIs
StateAccepted/In press - Jan 1 2018

Keywords

  • assay
  • fluorescence
  • GFP
  • 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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