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Mineralocorticoid Receptor Reporter Gene Assay – optimisation for medium to high throughput screening.


Cell based Nuclear receptor assays utilising reporters under the transcriptional control of an upstream activator sequence (UAS) and activated by the GAL4 DNA binding domain (DBD) expressed as a fusion protein with a Nuclear receptor ligand binding domain (LBD), are relatively simple to establish. These assays are universal in that all components apart from the LBD for the NR under investigation are identical. These assays are commonly used to profile compounds for agonist/antagonist properties with respect to potency and efficacy. The simplicity of the chimeric system make them robust for screening large sets of compounds. However they may not always reflect the activity of the compound in vivo. This activity may be better represented using reporter systems under the control of receptor specific response elements or promoters and endogenous or over-expressed full length receptors.

The chimeric system under investigation uses a Mineralocorticoid receptor (MR) LBD fused to Gal4 DBD and an UAS beta-lactamase reporter expressed in HEK293 cells. When agonist, Aldosterone, binds to the LBD, beta-lactamase is expressed. The full length receptor system uses a luciferase reporter under the control of MMTV LTR also expressed in HEK293 cells.

We have used an assay using transiently transfected U2-OS cells as our starting point and developed a384-well format low volume assay in HEK293S cells. Cells were transiently transfected, using MaxCyte transfection, with full-length MR encoding vector and a luciferase encoding vector. The key areas for assay optimisation were to determine the cell number and incubation time to establish a robust assay with a good assay window and low variability.
Using the more robust HEK293S cells an assay ready cell stock for cryo-preservation was successfully made, and by screening in low volume plates less reagents were consumed. A robust automation friendly assay showing comparable data to the original U2-OS assay was established.
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