Quantitation of transcription and clonal selection of single living cells with β-lactamase as reporter
Quantitation of transcription and clonal selection of single living cells with β-lactamase as reporter
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DOI：
10.1126/science.279.5347.84
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发表时间：
1998-01-02
期刊：
影响因子：
56.9
通讯作者：
Tsien, RY
中科院分区：
文献类型：
--
作者：
Zlokarnik, G;Negulescu, PA;Tsien, RY
在单个活着的哺乳动物细胞中，基因表达以β-内酰胺酶为报告基因，该酶以膜转换酯的形式降解细胞内负载的底物。每个酶分子通过破坏共振能量传递，使许多底物分子的荧光从绿色变为蓝色。这种波长漂移可以通过肉眼或彩色胶片在含有不到100个β-内酰胺酶分子的单个细胞中检测到。发射比的强劲变化揭示了实时基因表达的数量异质性，使流式细胞术能够进行克隆选择，并为在活的哺乳动物细胞中高通量筛选候选药物奠定了基础。
Gene expression was visualized in single living mammalian cells with beta-lactamase as a reporter that hydrolyzes a substrate loaded intracellularly as a membrane-permeant ester. Each enzyme molecule changed the fluorescence of many substrate molecules from green to blue by disrupting resonance energy transfer. This wavelength shift was detectable by eye or color film in individual cells containing less than 100 beta-lactamase molecules. The robust change in emission ratio reveals quantitative heterogeneity in real-time gene expression, enables clonal selection by flow cytometry, and forms a basis for high-throughput screening of pharmaceutical candidate drugs in living mammalian cells.