Online monitoring of stimulus-induced gene expression in pancreatic beta-cells

Fluorescent proteins have been extensively used as protein "tags" to study the subcellular localization of proteins and/or their translocation upon st imulation or as markers for transfection in transient and stable expression systems. However, they have not been frequently used as reporter genes to monitor stimulus-induced gene expression in mammalian cells. Here we demons trate the use of fluorescent proteins to study stimulus-induced gene transc ription. The general applicability of the approach is exemplified by doxycy clin-(Tet-On) and phorbol 12-myristate 13-acetate-induced (c-fos) promoter activation, with green fluorescent protein (GFP) and red fluorescent protei n (DsRed) as semiquantitative and immediate reporters, of transcription act ivation. Under the control of beta -cell-specific promoters, such as the ra t insulin 1 promoter or the rat upstream glucokinase promoter, this approac h allowed us to monitor online glucose-induced gene transcription in primar y p-cells at the single-cell level as well as in the context of the islet o f Langerhans. Applying discretely detectable fluorescent proteins, for exam ple GFP and DsRed, enabled us to simultaneously monitor stimulus-induced tr anscription by two different promoters in the same cell.