使用优化的MS2-MCP系统对哺乳动物细胞中的单个mRNA分子进行成像
Imaging Single mRNA Molecules in Mammalian Cells Using an Optimized MS2-MCP System.
作者信息
Vera Maria, Tutucci Evelina, Singer Robert H
机构信息
Department of Biochemistry, McGill University, Montreal, Canada.
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, USA.
出版信息
Methods Mol Biol. 2019;2038:3-20. doi: 10.1007/978-1-4939-9674-2_1.
Abstract
Visualization of single mRNAs in their native cellular environment provides key information to study gene expression regulation. This fundamental biological question triggered the development of the MS2-MCP (MS2-Capsid Protein) system to tag mRNAs and image their life cycle using widefield fluorescence microscopy. The last two decades have evolved toward improving the qualitative and quantitative characteristics of the MS2-MCP system. Here, we provide a protocol to use the latest versions, MS2V6 and MS2V7, to tag and visualize mRNAs in mammalian cells in culture. The motivation behind engineering MS2V6 and MS2V7 was to overcome a degradation caveat observed in S. cerevisiae with the previous MS2-MCP systems. While for yeast we recommend the use of MS2V6, we found that for live-cell imaging experiments in mammalian cells, the MS2V7 has improved reporter properties.
摘要
在天然细胞环境中对单个信使核糖核酸（mRNA）进行可视化，可为研究基因表达调控提供关键信息。这一基本生物学问题推动了MS2-MCP（MS2衣壳蛋白）系统的发展，该系统用于标记mRNA，并利用宽场荧光显微镜对其生命周期进行成像。在过去二十年中，人们致力于改进MS2-MCP系统的定性和定量特性。在此，我们提供了一个使用最新版本MS2V6和MS2V7在培养的哺乳动物细胞中标记和可视化mRNA的方案。设计MS2V6和MS2V7的目的是克服在酿酒酵母中使用先前的MS2-MCP系统时观察到的降解问题。虽然对于酵母我们建议使用MS2V6，但我们发现对于哺乳动物细胞的活细胞成像实验，MS2V7具有更好的报告特性。
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