Reporter Gene Assays

Initially, reporter gene assays have been one of the few really scalable cellular assays, which can be run in high-density plates [62, 63]. These assays monitor the transcriptional regulation of a promoter of the gene of interest linked to the coding region of a reporter gene. Using this principle, the activity of a cellular target and its subsequent signal transduction pathway will be linked to the expression of a readily detectable protein. Such proteins will mostly be enzymes, for example firefly and renilla luciferase, P-galactosidase, secreted alkaline phosphatase, chloramphenicol acetyltransferase, or P-lactamase. By coupling the response to the expression of an enzyme, a highly amplified - and therefore sensitive - signal is obtained [64, 65]. Alternatively, green fluorescent protein (GFP) can be used as signal [66]. Many drug targets will influence some transcriptional events in the cells, and are therefore amendable for reporter gene assays. Growth receptor pathways will, by definition, change transcriptional regulation, which is also achieved by nuclear receptors which are ligand-regulated transcription factors. G-protein coupled receptors (GPCRs) signaling can be monitored using cAMP-responsive element binding (CREB) or serum-responsive element (SRE) -activated promoter activity [67]. By using different constructs, a wide range of cellular events can be coupled to different read-outs via various substrates. Due to the broad application of the assay principle, only review articles are cited here. As a general disadvantage, reporter gene assays might suffer from interference of compounds acting distally to the target. Therefore, their results must be verified in a number of control assays aimed at filtering the non-target-related or cytotoxic effects [68].

138 | 5 Drug Screening Using Cell Lines: Cell Supply, High-Throughput and High-Content Assays 5.3.6

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