Lenstra Tineke L, Larson Daniel R
Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland.
Curr Protoc Mol Biol. 2016;113:14.24.1-14.24.15. doi: 10.1002/0471142727.mb1424s113. Epub 2016 Jan 4.
Visualization of single RNA molecules in living cells has enabled the study of synthesis, movement, and localization of mRNAs and has provided insight into gene regulation with sub-second temporal resolution and nanometer spatial resolution. Following transcription in single cells indicates that gene activity is heterogeneous between cells and also exhibits random variability over time even within single cells. Studies of mRNAs in yeast can take advantage of the powerful genetics available in this model organism and allow mechanistic questions to be addressed. In this chapter, we describe an approach for visualizing mRNA and transcription in live yeast cells. The method is based on binding of fluorescently labeled MS2 and PP7 coat proteins to stem loops sequences that are introduced into the gene of interest. We give detailed protocols for the construction of the necessary yeast strains, for image acquisition, and for validation.
活细胞中单个RNA分子的可视化，使得对mRNA的合成、移动和定位的研究成为可能，并为基因调控提供了亚秒级时间分辨率和纳米级空间分辨率的深入见解。单细胞转录过程的研究表明，基因活性在细胞间是异质的，并且即使在单个细胞内，随着时间的推移也表现出随机变异性。对酵母中mRNA的研究可以利用这种模式生物中强大的遗传学方法，并解决一些机制问题。在本章中，我们描述了一种在活酵母细胞中可视化mRNA和转录的方法。该方法基于荧光标记的MS2和PP7外壳蛋白与引入到感兴趣基因中的茎环序列的结合。我们给出了构建必要酵母菌株、图像采集和验证的详细方案。
