Imaging real-time gene expression in Mammalian cells with single-transcript resolution.
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA. Cold Spring Harbor Protocols
(Impact Factor: 4.63).
01/2007; 2007:pdb.prot4869. DOI: 10.1101/pdb.prot4869
INTRODUCTIONThe MS2 system provides optimal sensitivity for single-molecule detection in cells. It requires two genetically encoded moieties: a reporter mRNA that contains MS2 binding site (MBS) stem loops and a fluorescent MS2 coat protein (MCP-xFP) that binds to the stem loops with high affinity, thus tagging the mRNA within the cell. This protocol describes transfection of COS-7 cells with reporter RNA (e.g., pRSV-Z-24 MBS-β-actin) and MCP-xFP (e.g., pPolII-MCP-GFP-NLS) plasmids using calcium phosphate precipitation. The reporter mRNA plasmid must be co-transfected with the MCP-xFP-NLS plasmid for simultaneous expression in a cell. The unbound MCP-xFP-NLS is sequestered in the nucleus, leaving only the MCP-xFP-NLS that is bound to the reporter mRNA in the cytoplasm. This provides a high signal-to-noise ratio (SNR) that permits detection of single mRNA molecules. The Delta T Imaging System is used for image acquisition of fluorescent particles in the cells.
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