Imaging Single mRNA Dynamics in Live Neurons and Brains

Methods Enzymol. 2016;572:51-64. doi: 10.1016/bs.mie.2016.02.015. Epub 2016 Mar 22.

Abstract

RNA is a key player in the process of gene expression. Whereas fluorescence in situ hybridization allows single mRNA imaging in fixed cells, the MS2-GFP labeling technique enables the observation of mRNA dynamics in living cells. Recently, two genetically engineered mouse models have been developed for the application of the MS2-GFP system in live animals. First, the Actb-MBS mouse was generated by knocking in 24 repeats of the MS2 stem-loop sequence in the 3' untranslated region of the β-actin gene. Second, the MCP mouse was made to express the NLS-HA-MCP-GFP transgene in all cell types. By crossing Actb-MBS and MCP mice, a double homozygous mouse line, MCP×MBS, was established to visualize endogenous β-actin mRNA labeled with multiple green fluorescent proteins. By imaging hippocampal neurons or brain slices from MCP×MBS mice, the dynamics of mRNA, such as transcription, transport, and localization, can be studied at single mRNA resolution. In this chapter, we explain the basics of MCP×MBS mice and describe methods for utilizing these animals.

Keywords: Brain slice; MS2-GFP; Neuron; Single-molecule imaging; Transcription; Two-photon microscopy; mRNA; mRNA localization; β-Actin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions
  • Actins / genetics
  • Animals
  • Brain / cytology*
  • Brain / metabolism
  • Capsid Proteins / genetics
  • Cell Culture Techniques / methods
  • Cell Survival
  • Cells, Cultured
  • Genetic Engineering
  • Green Fluorescent Proteins / genetics
  • Levivirus / genetics
  • Mice
  • Microscopy, Confocal / methods
  • Microtomy / methods
  • Molecular Imaging / methods*
  • Neurons / cytology*
  • Neurons / metabolism
  • RNA, Messenger / analysis*
  • RNA, Messenger / genetics
  • Staining and Labeling / methods
  • Transcription, Genetic

Substances

  • 3' Untranslated Regions
  • Actins
  • Capsid Proteins
  • RNA, Messenger
  • Green Fluorescent Proteins