Bensidoun Pierre, Verbrugghe Morgane, Lagha Mounia
Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535, University of Montpellier, Montpellier, France.
Methods Mol Biol. 2025;2923:215-229. doi: 10.1007/978-1-0716-4522-2_13.
The ultimate output of gene expression is to ensure that proteins are synthesized at the right levels, locations, and timings. Recently different imaging-based methods have been developed to visualize the translation of single mRNA molecules. These methods rely on signal amplification with the introduction of an array of a short peptide sequence (a tag such as SunTag), recognized by a genetically encodable single-chain antibody (a detector such as scFv). In this chapter, we discuss such methods to image and quantify translation dynamics in the early Drosophila embryo and provide examples based on a twist-32XSunTag reporter. We outline a step-by-step protocol to light-up translation in living embryos. We also detail a combinatorial strategy in fixed samples (smFISH-IF), allowing to distinguish single mRNA molecules engaged in translation.
基因表达的最终输出是确保蛋白质在正确的水平、位置和时间合成。最近，已经开发出了不同的基于成像的方法来可视化单个mRNA分子的翻译过程。这些方法依赖于通过引入一系列短肽序列（如SunTag这样的标签）进行信号放大，该短肽序列可被基因编码的单链抗体（如scFv这样的检测器）识别。在本章中，我们将讨论此类用于成像和量化果蝇早期胚胎中翻译动力学的方法，并给出基于twist-32XSunTag报告基因的示例。我们概述了在活胚胎中开启翻译的分步方案。我们还详细介绍了固定样本中的组合策略（smFISH-IF），该策略能够区分参与翻译的单个mRNA分子。
