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 stimulation 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 demonstrate the use of fluorescent proteins to study stimulus-induced gene transcription. The general applicability of the approach is exemplified by doxycyclin-(Tet-On) and phorbol 12-myristate 13-acetate-induced (c-fos) promoter activation, with green fluorescent protein (GFP) and red fluorescent protein (DsRed) as semiquantitative and immediate reporters, of transcription activation. Under the control of beta-cell-specific promoters, such as the rat insulin 1 promoter or the rat upstream glucokinase promoter, this approach allowed us to monitor online glucose-induced gene transcription in primary beta-cells at the single-cell level as well as in the context of the islet of Langerhans. Applying discretely detectable fluorescent proteins, for example GFP and DsRed, enabled us to simultaneously monitor stimulus-induced transcription by two different promoters in the same cell.
Department of Molecular Medicine, Rolf Luft Center for Diabetes Research, Karolinska Institute, Stockholm, Sweden.
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D000818:Animals; D002460:Cell Line; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D005947:Glucose / Q000494:pharmacology*; D049452:Green Fluorescent Proteins; D007091:Image Processing, Computer-Assisted; D007328:Insulin / Q000494:pharmacology*; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008164:Luminescent Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
D007328:Insulin; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005947:Glucose