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.
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Acknowledgments
The authors are grateful to Xihua Meng and Lydia Tesfa (Grant P30CA013330) for technical help. Support was provided by National Institutes of Health Grants R01GM057071 to R.H.S. and R21AG055083 to M.V. and by the Swiss National Science Foundation for Fellowships P2GEP3_155692 and P300PA_164717 to E.T.
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Vera, M., Tutucci, E., Singer, R.H. (2019). Imaging Single mRNA Molecules in Mammalian Cells Using an Optimized MS2-MCP System. In: Shav-Tal, Y. (eds) Imaging Gene Expression. Methods in Molecular Biology, vol 2038. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9674-2_1
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DOI: https://doi.org/10.1007/978-1-4939-9674-2_1
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