Visualization of Single mRNAs in Live Neurons

Jae Youn Shim, Byung Hun Lee, Hye Yoon Park

Research output: Chapter in Book/Report/Conference proceedingChapter

2 Scopus citations

Abstract

Transcription and post-transcriptional regulations are critical in gene expression. To study the spatiotemporal regulation of RNA inside a cell, techniques for high-resolution imaging of RNA have been developed. In this chapter, we describe RNA fluorescent labeling methods using MS2 and PP7 systems to detect single RNA molecules in live neurons. We use hippocampal neurons cultured from knock-in mouse models in which β-actin or Arc mRNAs are tagged with MS2 or PP7 stem-loops. Adeno-associated virus (AAV) or lentiviral vectors are used to express MS2 or PP7 capsid proteins fused with GFP in those neurons. Then, GFP-labeled RNAs in live neurons can be detected by epifluorescence microscopy, and their moving pathways can be analyzed using single-particle tracking software. For these processes, we introduce protocols for neuron culture, transfection, imaging, and particle tracking methods.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages47-61
Number of pages15
DOIs
StatePublished - 2019
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume2038
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Bibliographical note

Funding Information:
This work was supported by the Creative-Pioneering Researchers Program through Seoul National University.

Publisher Copyright:
© Springer Science+Business Media, LLC, part of Springer Nature 2019.

Keywords

  • Arc
  • Endogenous mRNA
  • MS2-GFP
  • Neuron
  • PP7-GFP
  • Single-molecule imaging
  • β-Actin

Fingerprint

Dive into the research topics of 'Visualization of Single mRNAs in Live Neurons'. Together they form a unique fingerprint.

Cite this