GR-related Assays (Glucocorticoid)
luciferase reporter constructs validated for glucocorticoid receptor
The LightSwitch™ Validated Glucocorticoid Receptor (GR) Pathway Collection is a comprehensive set of sequence-verified, transfection-ready, promoter reporter constructs that can be used to accurately quantify transcriptional activation and repression in response to induction of pathways regulated by the glucocorticoid receptor. These constructs are a subset of the 18,000-member LightSwitch Promoter Reporter GoClone™ Collection that were selected based on sequence motif analysis, published information and in-house analysis of each promoter's activity* using our LightSwitch Luciferase Assay.
The GR Biomarker Set contains 8 of the highest responding promoters under our chosen test conditions, as well as the promoters of 2 housekeeping genes (Figure 1). The GR Profiling Plate contains 88 promoter reporter constructs that showed significant activation or repression in response to induction (See the GR Profiling Plate tab below for data and a list of constructs). Active Motif also offers a GR Pathway Reporter Stable Cell Line to facilitate high-throughput screening of the glucocorticoid receptor pathway.
Figure 1: Induction ratios for each member of the GR Biomarker Set.
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Glucocorticoid Receptor (GR) Pathway
Nuclear receptors, ligand-activated transcription factors, are critical for understanding gene regulation and the genomic events that lead to development, differentiation and growth in an organism. Ligands for nuclear receptors (NRs) include a variety of small molecules such as steroids and fatty acids. Xenobiotics and various pharmaceutical agents also serve as NR ligands, illustrating the importance of NR-mediated toxicity screening.
Glucocorticoids play an essential role in maintaining basal and stress-related homeostasis. Glucocorticoid Receptor, also known as nuclear receptor subfamily 3, group C, member 1 (NR3C1), can act either as a transcription factor by binding glucocorticoid response elements (GREs), or as a regulator of other transcription factors. GR is found in the cytoplasm in the absence of ligand binding, but once a ligand has bound, a conformational change induces transport to the nucleus. GR mutations can result in glucocorticoid resistance or hypersensitivity, pathologic alterations of metabolism and excessive or suppressed immune response.
The collection of validated LightSwitch GR-related promoter reporters enables you to:
- Understand the mechanisms by which GR regulated genes are induced or repressed
- Quantify the functional consequences of transcription factor binding – while many other technologies give a qualitative view of transcription factor level, our promoter reporter assays quantify the effects of transcription factor binding
- Confirm data from ChIP, ChIP-chip or ChIP-Seq experiments
- Measure the effect of sequence variants and mutagenesis on promoter function
- Screen for promoter activation or develop activity profiles across the GR signaling pathway for many compounds or conditions in parallel
LightSwitch Luciferase Assay System
The LightSwitch Luciferase Assay System is a complete solution for studying transcriptional & post-transcriptional gene regulation in living mammalian cells. It enables you to directly measure the functional activity of promoters and 3´UTRs through use of an engineered luciferase gene (RenSP) and optimized assay & transfection reagents that ensure superior, reproducible results. In addition to Validated Pathway Collections, the LightSwitch System includes collections of over 30,000 cloned human promoter reporter constructs and human 3´UTR reporter constructs, miRNA mimics and inhibitors, stable cell lines, as well as collections of synthetic long-range response element constructs and synthetic 3´UTRs target validation constructs that can provide more sensitivity than endogenous sequences. Comprehensive Custom Services are also available
LightSwitch Promoter Reporter Assays.
Advantages of the LightSwitch Luciferase Assay System
- Quantitative – Novel RenSP luciferase technology allows you to measure promoter activity with industry-leading sensitivity and dynamic range.
- Simple, fast, complete solution – With pre-cloned LightSwitch Reporter vectors and optimized transfection & assay reagents, you can study regulation of your gene today. No cloning, DNA preparation or optimization is needed, and most studies do not require any internal transfection controls.
- Comprehensive and verified – The genome-wide LightSwitch Reporter Collections are sequence-verified, transfection-ready promoter and 3´UTR reporter vectors.
- Functionally insightful – Learn about the actual effects of transcription factor binding to verify computational predictions and supplement microarray or sequencing data.
- Cost-effective – Efficiently screen for activation and/or repression using a multitude of conditions.
LightSwitch™ GR Pathway Profiling Plate
The GR Profiling Plate is a subset of promoter reporter constructs chosen from the LightSwitch Promoter Reporter GoClone™ Collection. Promoters that were expected to be up- or down-regulated in the glucocorticoid receptor pathway were chosen based on motif analysis and published information. In-house luciferase testing was then performed to select those promoter reporter constructs that were activated or repressed in response to our induction methods, which were to treat HT1080 cells with 100 nM dexamethasone, with 1 µM prednisone, or with 1 µM cortisone for 4 hours following transfection.
GR Profiling Plate heatmap
The heatmap at right shows the untreated and induced activity for all constructs that are members of the pathway profiling plate. On the right side of the figure, the colors represent log2 transformed ratios of treated/untreated activity according to the color scale shown at the base of the heatmap. Black indicates no change; the intensity of red or blue color indicates levels of induction or repression, respectively. On the left side of the figure, the intensity of yellow color depicts the activity of each promoter in the unstimulated (untreated) condition. All untreated signals are relative to the highest and lowest values within the individual experiment. (“DEX” = HT1080 fibrosarcoma cells treated with 100 nM dexamethasone for 4 hours; “PRED” = HT1080 fibrosarcoma cells treated with 1 µM prednisone for 4 hours ; “CORT” = HT1080 fibrosarcoma cells treated with 1 µM cortisone for 4 hours.)
GR Profiling Plate contents
The GR Profiling Plate includes 88 promoter reporter constructs (including the 8 top-responding constructs that comprise the GR Biomarker Set, as well as 6 housekeeping / random promoters that are unlikely to respond to GR induction conditions). The controls can be used to correct for non-specific changes in overall signal in response to the stimulus. For example, some compounds used for induction are very toxic and result in a significant decrease in overall luciferase signal across the entire panel of constructs in a non-promoter-specific manner. Also included in the plate are 8 transfection controls that are used to determine the conditions that yield maximum transfection efficiency and reproducibility.
If you would like to order 5 or fewer members of the GR Profiling Plate, you can use the Product IDs found in the file for the plate map above to order directly on Active Motif's SwitchGear Genomics website. If you wish to order the entire plate (or a minimum of 5 or more members), click here to send us an e-mail detailing which constructs you would like, and what amount (5 µg, 10 µg, 1 mg, etc.). We will send you a quote based on your request.
Click image to enlarge.
The GR Biomarker Set contains 5 µg aliquots each of 8 promoter reporter constructs and 2 housekeeping promoter constructs (NUB1 & SLC37A3) for use as controls. Click on the links below for clone information.
The GR Profiling Plate includes 88 promoter reporter constructs and 8 transfection control constructs (4 constructs from housekeeping genes with promoter activities of varying strengths (P1-P4), and 4 constructs with random 1 kb fragments (R1-R4)). To download a plate map with links to clone information, please click here.