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Visualization of Single mRNAs in Live Neurons

  • Jae Youn Shim
  • Byung Hun Lee
  • Hye Yoon ParkEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2038)

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.

Key words

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

Notes

Acknowledgments

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

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Jae Youn Shim
    • 1
  • Byung Hun Lee
    • 1
  • Hye Yoon Park
    • 1
    • 2
    • 3
    Email author
  1. 1.Department of Physics and AstronomySeoul National UniversitySeoulSouth Korea
  2. 2.The Institute of Molecular Biology and GeneticsSeoul National UniversitySeoulSouth Korea
  3. 3.The Institute of Applied PhysicsSeoul National UniversitySeoulSouth Korea

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