Skirball Institute of Biomolecular Medicine, Department of Cell Biology, NYU Grossman School of Medicine, New York, NY 10016, USA.
Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Canada.
STAR Protoc. 2020 Sep 24;1(3):100112. doi: 10.1016/j.xpro.2020.100112. eCollection 2020 Dec 18.
Advances in imaging technologies, gene editing, and fluorescent molecule development have made real-time imaging of nucleic acids practical. Here, we detail methods for imaging the human telomerase RNA template, hTR via the use of three inserted MS2 stem loops and cognate MS2 coat protein (MCP) tagged with superfolder GFP or photoactivatable GFP. These technologies enable tracking of the dynamics of RNA species through Cajal bodies and offer insight into their residence time in Cajal bodies through photobleaching and photoactivation experiments. For complete details on the use and execution of this protocol, please refer to Laprade et al. (2020).
成像技术、基因编辑和荧光分子的发展使得实时成像核酸成为可能。在这里，我们详细介绍了通过使用三个插入的 MS2 茎环和带有超折叠 GFP 或光激活 GFP 的同源 MS2 外壳蛋白 (MCP) 对人端粒酶 RNA 模板 hTR 进行成像的方法。这些技术能够通过 Cajal 体跟踪 RNA 种类的动态，并通过光漂白和光激活实验深入了解它们在 Cajal 体中的停留时间。有关该方案使用和执行的完整详细信息，请参阅 Laprade 等人（2020 年）。
