Abstract
Live-cell single mRNA imaging is a powerful tool but has been restricted in higher eukaryotes to artificial cell lines and reporter genes. We describe an approach that enables live-cell imaging of single endogenous labeled mRNA molecules transcribed in primary mammalian cells and tissue. We generated a knock-in mouse line with an MS2 binding site (MBS) cassette targeted to the 3′ untranslated region of the essential ββ-actin gene. As β-actin–MBS was ubiquitously expressed, we could uniquely address endogenous mRNA regulation in any tissue or cell type. We simultaneously followed transcription from the β-actin alleles in real time and observed transcriptional bursting in response to serum stimulation with precise temporal resolution. We tracked single endogenous labeled mRNA particles being transported in primary hippocampal neurons. The MBS cassette also enabled high-sensitivity fluorescence in situ hybridization (FISH), allowing detection and localization of single β-actin mRNA molecules in various mouse tissues.
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Acknowledgements
We thank D. Ron (Skirball Institute of Biomolecular Medicine) for providing the SV40 large T antigen plasmid (pBSSVD2005) and the MEF immortalization protocol, C. Chaponnier (Université de Genève) for the gift of antibodies, C. Montagna for assistance in karyotyping and R.S. Sellers for assistance in tissue examination. The phage-UbC RIG vector for lentiviral expression was a generous gift from G. Mostoslavsky and G. Vainer (Harvard University). Microscopy equipment for the live-cell imaging experiments was provided by the Gruss Lipper Biophotonics Center. T.L. was supported by a Human Frontier Science Program long-term fellowship. This work was supported by US National Institutes of Health (GM84364, 86217 and EB2060 to R.H.S.), the Jane Stern Lebell Family Fellowship of Bar-Ilan University to Y.S.-T., and US-Israel Binational Science Foundation to Y.S.-T. and R.H.S. H.Y.P. is supported by National Research Service awards (F32-GM087122). A.L.W. is supported by a development grant from the Muscular Dystrophy Association (MDA68802).
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Contributions
T.L. performed the biochemistry experiments, the tissue FISH imaging, serum response live-cell imaging, and quantitative mRNA FISH, analyzed the data and wrote the paper. K.C. generated cell lines and performed the neuron live-cell imaging. X.D. and Y.S.-T. generated the mouse line. A.L.W. performed FISH mRNA localization experiments. J.A.C. performed the serum response live-cell imaging. H.Y.P. performed the live-cell localization experiments. V.d.T. generated cell lines. M.L.-J. performed the biochemistry experiments. R.H.S. consulted on the research and helped write the paper.
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Supplementary Text and Figures
Supplementary Figures 1–8, Supplementary Note 1 (PDF 1175 kb)
Supplementary Video 1
Live-cell imaging of a serum-induced primary fibroblast. MCP-GFP-labeled mRNA particles can be detected moving around the cell; the two transcription sites of the primary cell appear as two bright spots in the nucleus. Note that the transcription sites intensity is intentionally saturated to allow visualizing the dimmer single particles. (MOV 1158 kb)
Supplementary Video 2
Individual mRNP moving along a neuronal process. (MOV 2377 kb)
Supplementary Video 3
Individual mRNP moving along a neuronal process. The process is the same as the one imaged in Supplementary Video 2. (MOV 2138 kb)
Supplementary Video 4
Anterograde mRNP motion along a neuronal process. (MOV 232 kb)
Supplementary Video 5
Retrograde mRNP motion along a neuronal process. (MOV 211 kb)
Supplementary Video 6
Bidirectional mRNP motion along a neuronal process. (MOV 2251 kb)
Supplementary Video 7
Branching mRNP motion along a neuronal process. (MOV 2024 kb)
Supplementary Video 8
Immobile mRNP in a neuronal process. (MOV 926 kb)
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Lionnet, T., Czaplinski, K., Darzacq, X. et al. A transgenic mouse for in vivo detection of endogenous labeled mRNA. Nat Methods 8, 165–170 (2011). https://doi.org/10.1038/nmeth.1551
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DOI: https://doi.org/10.1038/nmeth.1551
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