Cite as: Cold Spring Harb. Protoc.; 2007; doi:10.1101/pdb.prot4869

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protocolProtocol

Imaging Real-Time Gene Expression in Mammalian Cells with Single-Transcript Resolution

Amber L. Wells1, John S. Condeelis1,2, Robert H. Singer1,2,3, and Daniel Zenklusen1

1 Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
2 Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA

3Corresponding author (rhsinger{at}aecom.yu.edu)


INTRODUCTION

The 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-ß-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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