Shigeoka Toshiaki, Jung Jane, Holt Christine E, Jung Hosung
Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge, CB2 3DY, UK.
Department of Anatomy, Brain Research Institute, and Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea.
Methods Mol Biol. 2018;1649:85-94. doi: 10.1007/978-1-4939-7213-5_5.
Translating ribosome affinity purification (TRAP) is a widely used technique to analyze ribosome-bound mRNAs in particular target cells that express a tagged ribosomal protein. We developed axon-TRAP-RiboTag, a TRAP-based method that allows purification and identification of translated mRNAs from distal neuronal axons in mouse, and identified more than 2000 of translated mRNAs in retinal ganglion cell (RGC) axons in vivo. The use of Cre-negative littermate control to filter out false-positive signals allows unbiased detection, and combining TRAP with in vitro ribosome run-off enables identification of actively translated mRNAs. Here, we describe a detailed protocol to identify translated mRNAs in RGC axons in mouse in vivo. This method can be applied to any neurons whose cell bodies and distal axons are anatomically separated.
翻译核糖体亲和纯化（TRAP）是一种广泛应用的技术，用于分析在表达标记核糖体蛋白的特定靶细胞中与核糖体结合的mRNA。我们开发了轴突TRAP-RiboTag，这是一种基于TRAP的方法，可用于从小鼠远端神经元轴突中纯化和鉴定翻译后的mRNA，并在体内视网膜神经节细胞（RGC）轴突中鉴定出2000多种翻译后的mRNA。使用Cre阴性同窝对照来过滤假阳性信号可实现无偏检测，将TRAP与体外核糖体延伸相结合能够鉴定出活跃翻译的mRNA。在这里，我们描述了一种在小鼠体内鉴定RGC轴突中翻译后mRNA的详细方案。该方法可应用于任何细胞体和远端轴突在解剖学上分离的神经元。
