Service Description
Provider: INDIGO Biosciences, Inc. provides Nuclear Receptor Reporter Assay based products and services for drug discovery and toxicology. INDIGO specializes in the development of frozen, whole-cell Nuclear Receptor Assay Systems for use in small compound HTS, off-target drug interactions, and toxicology screens.
Background: Retinoic acid receptors (RARs) are nuclear hormone receptors of the NRB1 class, which function as heterodimers with retinoid X receptors (RXRs). There are three distinct RAR subtypes; RARalpha, RARbeta and RARgamma. RARalpha is present in most tissue types, whereas RARbeta and RARgamma expression is more selective. RXR-RAR heterodimers act as ligand-dependent transcriptional regulators by binding to the specific retinoic acid response element (RARE) found in the promoter regions of target genes. In the absence of an RAR agonist, RXR-RAR recruits co-repressor proteins such as NCoR and associated factors such as histone deacetylase to maintain a condensed chromatin structure. RAR agonist binding stimulates co-repressor release and co-activator complexes, such as histone acetyltransferase, are recruited to activate transcription. RARs transduce retinoid signals in vivo, which mediates proper embryogenesis, differentiation and growth arrest. Specifically, RXRalpha-RARgamma heterodimers are necessary for growth arrest and viseral and primitive endodermal differentiation, whereas RXRalpha-RARalpha is required for cAMP-dependent parietal endodermal differentiation. In vitro it has been difficult to elucidate the roles of individual subtypes as functional RAR knockouts generate artificial redundancies that are thought not to exist under normal conditions.
Service Details: This assay can be utilized to examine the agonism (activation) or antagonism (inhibition of an agonist) on the nuclear receptor. Mammalian cells are transfected with the ligand binding domain of the NR fused to a heterologous DNA binding domain as well as the response element for this DNA binding motif linked to a reporter gene. A transfection efficiency control plasmid is also included. These transfected cells are seeded in 96-well plates and then treated overnight with test compound at 37°C. Cells are then lysed and NR activation reporter activity as well as transfection efficiency reporter activity is assessed. A positive control compound (6 concentrations in triplicate) as well as vehicle control (triplicate) is included in every experiment.
For dose response curves, it is suggested that at least six concentrations of the test compounds be examined, covering a wide range of dilutions (3 log-units). In addition, we recommend that each compound/concentration be assayed in triplicate.
Deliverable: Downloadable data report containing raw and analyzed data. EC50 and peak activity will be determined using nonlinear regression. Dose-response data will be plotted for the test compounds as well as the positive control. A sample data report is available.
Price: The price of the assay includes a set-up fee, as well as a per well charge. Discounts are provided for academicians and for larger projects. Please contact us by email or phone (814) 234-1919 Ext. 303 for a price quote.
Sample Submission: The minimum sample requirement is 25 µl of a 10 mM stock solution, with the preferred sample submission of 100 mM stock solution in DMSO. A sample submission sheet is available.
Species available: Reporter assays for human are optimized and ready for use. Assays for other species can be custom prepared. Please visit the Custom Assay Development page for more details
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