Mandic A, Strebinger D, Regali C, Phillips N E, Suter D M
The Institute of Bioengineering (IBI), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), 1015 Lausanne, Switzerland.
The Institute of Bioengineering (IBI), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), 1015 Lausanne, Switzerland.
Methods. 2017 May 1;120:65-75. doi: 10.1016/j.ymeth.2017.04.008. Epub 2017 Apr 27.
Gene expression is at the heart of virtually any biological process, and its deregulation is at the source of numerous pathological conditions. While impressive progress has been made in genome-wide measurements of mRNA and protein expression levels, it is still challenging to obtain highly quantitative measurements in single living cells. Here we describe a novel approach based on internal tagging of endogenous proteins with a reporter allowing luminescence and fluorescence time-lapse microscopy. Using luminescence microscopy, fluctuations of protein expression levels can be monitored in single living cells with high sensitivity and temporal resolution over extended time periods. The integrated protein decay reporter allows measuring protein degradation rates in the absence of protein synthesis inhibitors, and in combination with absolute protein levels allows determining absolute amounts of proteins synthesized over the cell cycle. Finally, the internal tag can be excised by inducible expression of Cre recombinase, which enables to estimate endogenous mRNA half-lives. Our method thus opens new avenues in quantitative analysis of gene expression in single living cells.
基因表达几乎是任何生物过程的核心，其失调是众多病理状况的根源。尽管在全基因组范围内测量mRNA和蛋白质表达水平方面已取得了令人瞩目的进展，但在单个活细胞中获得高度定量的测量结果仍然具有挑战性。在此，我们描述了一种基于用报告基因对内源蛋白质进行内部标记的新方法，该报告基因可实现发光和荧光延时显微镜观察。使用发光显微镜，可以在单个活细胞中长时间以高灵敏度和时间分辨率监测蛋白质表达水平的波动。整合的蛋白质降解报告基因能够在不存在蛋白质合成抑制剂的情况下测量蛋白质降解速率，并且与绝对蛋白质水平相结合，可以确定在细胞周期中合成的蛋白质的绝对量。最后，可通过诱导表达Cre重组酶切除内部标签，这使得能够估计内源性mRNA的半衰期。因此，我们的方法为单个活细胞中基因表达的定量分析开辟了新途径。
