Figure 1

Imaging transcription from a single allele. (a) The principle underlying the MS2 mRNA reporter system. RNA polymerases (brown) initiating transcription from the promoter (blue) progress along the reporter gene (green). Upon reaching the cassette of MBS sequences (orange), each MBS sequence (usually 24 in all) forms a stem-loop in the nascent mRNA. Coexpressed fluorescently tagged MCP protein (MCP-GFP) binds the stem-loops, resulting in a fluorescent mRNA particle that can be detected by fluorescence microscopy. (b) Insertion of the MS2 construct into host cells by site-specific homologous recombination. A plasmid containing a promoter-gene construct (blue/green) and an MBS cassette (orange) upstream of the recombination target sequence (FRT, red triangle) is transfected into a cell line that harbors a single FRT sequence (red triangle) within its genome. Homologous recombination mediated by coexpression of the FLP recombinase (not shown) results in the integration of the reporter gene at that FRT site. CCND1 promoter and reporter sequences are shown here, but any combination of promoter and reporter sequence could be used.