Institut de Génétique Moléculaire de Montpellier, University of Montpellier, CNRS, 34293 Montpellier, France.
Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, 61231 Bad Nauheim, Germany.
RNA. 2024 Sep 16;30(10):1374-1394. doi: 10.1261/rna.080140.124.
Live imaging of translation based on tag recognition by a single-chain antibody is a powerful technique to assess translation regulation in living cells. However, this approach is challenging and requires optimization in terms of expression level and detection sensitivity of the system, especially in a multicellular organism. Here, we improved existing fluorescent tools and developed new ones to image and quantify nascent translation in the living embryo and in mammalian cells. We tested and characterized five different green fluorescent protein variants fused to the single-chain fragment variable (scFv) and uncovered photobleaching, aggregation, and intensity disparities. Using different strengths of germline and somatic drivers, we determined that the availability of the scFv is critical in order to detect translation throughout development. We introduced a new translation imaging method based on a nanobody/tag system named ALFA-array, allowing the sensitive and simultaneous detection of the translation of several distinct mRNA species. Finally, we developed a largely improved RNA imaging system based on an MCP-tdStaygold fusion.
基于单链抗体标签识别的翻译的实时成像技术是评估活细胞中翻译调控的一种强大技术。然而，这种方法具有挑战性，需要对系统的表达水平和检测灵敏度进行优化，特别是在多细胞生物中。在这里，我们改进了现有的荧光工具，并开发了新的工具，以对活体胚胎和哺乳动物细胞中的新生翻译进行成像和定量。我们测试和表征了融合到单链片段可变区 (scFv) 的五种不同的绿色荧光蛋白变体，并揭示了荧光漂白、聚集和强度差异。利用不同强度的种系和体细胞驱动子，我们确定 scFv 的可用性对于在整个发育过程中检测翻译至关重要。我们引入了一种基于纳米体/标签系统的新翻译成像方法，命名为 ALFA-array，它可以灵敏且同时检测几种不同 mRNA 种类的翻译。最后，我们开发了一种基于 MCP-tdStaygold 融合的大大改进的 RNA 成像系统。
