Imaging Single mRNA Molecules in Mammalian Cells Using an Optimized MS2-MCP System

Methods Mol Biol. 2019;2038:3-20. doi: 10.1007/978-1-4939-9674-2_1.

Abstract

Visualization of single mRNAs in their native cellular environment provides key information to study gene expression regulation. This fundamental biological question triggered the development of the MS2-MCP (MS2-Capsid Protein) system to tag mRNAs and image their life cycle using widefield fluorescence microscopy. The last two decades have evolved toward improving the qualitative and quantitative characteristics of the MS2-MCP system. Here, we provide a protocol to use the latest versions, MS2V6 and MS2V7, to tag and visualize mRNAs in mammalian cells in culture. The motivation behind engineering MS2V6 and MS2V7 was to overcome a degradation caveat observed in S. cerevisiae with the previous MS2-MCP systems. While for yeast we recommend the use of MS2V6, we found that for live-cell imaging experiments in mammalian cells, the MS2V7 has improved reporter properties.

Keywords: MS2 system; MS2V6; MS2V7; Quantitative fluorescence microscopy; Short-lived mRNAs; Single cell; Single-molecule imaging; smFISH.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Aptamers, Nucleotide / genetics
  • Capsid Proteins / genetics
  • Capsid Proteins / metabolism*
  • Cell Line
  • Gene Expression Regulation, Fungal
  • Humans
  • In Situ Hybridization, Fluorescence*
  • Levivirus / genetics
  • Levivirus / metabolism*
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Microscopy, Fluorescence*
  • Molecular Imaging / methods*
  • RNA Stability
  • RNA, Fungal / genetics
  • RNA, Fungal / metabolism*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Single Molecule Imaging / methods*
  • Time Factors

Substances

  • Aptamers, Nucleotide
  • Capsid Proteins
  • Luminescent Proteins
  • RNA, Fungal
  • RNA, Messenger
  • Recombinant Fusion Proteins