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Axon-TRAP-RiboTag: Affinity Purification of Translated mRNAs from Neuronal Axons in Mouse In Vivo.

Authors
 Toshiaki Shigeoka  ;  Jane Jung  ;  Christine E. Holt  ;  Hosung Jung 
Citation
 Methods in Molecular Biology (Clifton, N.J.), Vol.1649 : 85-94, 2018 
Journal Title
 Methods in Molecular Biology (Clifton, N.J.) 
ISSN
 1064-3745 
Issue Date
2018
MeSH
Animals ; Axons/metabolism* ; Breeding ; Chromatography, Affinity/methods* ; Immunoprecipitation ; Mice ; Protein Biosynthesis/genetics* ; RNA/isolation & purification ; Ribosomes/metabolism*
Keywords
Axon ; Immunoprecipitation ; Neuron ; Ribosome ; Translation ; mRNAs
Abstract
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.
Full Text
https://link.springer.com/protocol/10.1007%2F978-1-4939-7213-5_5
DOI
10.1007/978-1-4939-7213-5_5
Appears in Collections:
1. College of Medicine (의과대학) > Dept. of Anatomy (해부학교실) > 1. Journal Papers
Yonsei Authors
Jung, Ho Sung(정호성) ORCID logo https://orcid.org/0000-0002-5059-8050
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/165261
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