A Rapid and Precise Mutation-Activated Fluorescence Reporter for Analyzing Acute Mutagenesis Frequency.,Cell Chemical Biology

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A Rapid and Precise Mutation-Activated Fluorescence Reporter for Analyzing Acute Mutagenesis Frequency.
Cell Chemical Biology ( IF 6.6 ) Pub Date : 2018-06-14 , DOI: 10.1016/j.chembiol.2018.05.010
Michael D Birnbaum ₁ , Leah Nemzow ₂ , Akhilesh Kumar ₁ , Feng Gong ₃ , Fangliang Zhang ₄

Affiliation

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.

中文翻译：

一种快速、精确的突变激活荧光报告基因，用于分析急性突变频率。

诱变报告基因对于量化基因组稳定性至关重要。然而，当前的方法依赖于细胞存活/死亡来报告突变，这需要数周时间并且无法评估急性或时间依赖性变化。现有方法还存在其他限制，例如细胞类型限制。利用我们发现 mCherryFP 荧光依赖于残基 Trp98，我们用终止密码子替换该密码子以生成突变生物传感器（称为 CherryOFF），并以绿色荧光蛋白 (GFP) 作为内部对照。我们发现该生物传感器的红色荧光是由特定的 A/TG/C 核苷酸转换激活的。与已建立的次黄嘌呤磷酸核糖转移酶测定相比，我们的报告器具有相似或更好的检测物理/化学诱变剂或突变相关基因操作引起的突变频率变化的能力。此外，CherryOFF-GFP 可以独立于细胞死亡事件报告突变，可以适应多种细胞类型，并且可以在 1 天内生成读数以测量急性或时间依赖性事件。

更新日期：2018-08-17

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