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 3-2 untranslated region of the essential β-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.
Bibliographical noteFunding Information:
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).