Reporter gene assay
Two methods are mainly used to search for ligands of nuclear receptors: a binding assay using isolated and purified nuclear receptors, and a reporter gene assay using recombinant cells.
As described above, nuclear receptors are transcription factors that function through three steps: 1. ligand binding, 2. binding to specific DNA sequences, and 3. transcriptional activation of downstream genes. Since binding assays only evaluate the binding between the nuclear receptor and the ligand, they can evaluate 1. ligand binding but not transcriptional activity. Potential ligands of nuclear receptors must be able to pass easily through cell membranes. However, the process of intracellular uptake cannot be evaluated by a binding assay. In some cases, even when a binding assay shows that a substance binds to a nuclear receptor, the substance cannot function as a ligand because it has poor cell permeability.
A reporter gene assay can provide more reliable initial data than a binding assay, because the former uses cells to evaluate these three steps comprehensively.
The structure of reporter gene assays
Plasmid 1 carries a nuclear receptor sequence that produces a nuclear receptor protein within the cell. In this nuclear receptor, the LBD is expressed from its original sequence; however, the DBD is expressed as a recombinant fusion protein that binds to a DNA sequence called GAL4.
Plasmid 2 carries GAL4 and luciferase sequences. The fusion protein expressed from Plasmid 1 binds to this GAL4, causing expression of the downstream luciferase gene.
When energy (ATP) and substrate are subsequently added, they react with the luciferase, causing emission of light. This light emission is measured to determine the transcriptional activity of the luciferase gene resulting from the binding of the ligand to the nuclear receptor.
In summary, a nuclear receptor and its target gene are artificially introduced into a single type of cell under specific conditions in order to measure the binding of the ligand to the nuclear receptor and the resulting transcriptional activity.
In each experiment, since a plasmid is transiently introduced (transfected) into a cell, an internal standard plasmid is simultaneously introduced to standardize transfection efficiency. In addition, the types, passage numbers, etc. of the cells to be used are strictly controlled in order to eliminate all possible factors that affect measurement.
Experimental system
We will verify that the concentrations of received samples are non-toxic, dilute them to three concentrations, and measure their luminescence (n=3). The number of concentrations and repetitive measurements can be changed as needed.
Other services
*Detection of mRNA expression by real time PCR
In reporter gene assays, luciferase is used as an indicator to simulate gene expression. In actual cells and tissues, target gene expression can be detected by measurement of mRNA expression levels. For the latter kind of detection, cells that have target nuclear receptors are selected. After the sample is introduced into the cells, RNA is collected in order to measure the mRNA expression levels of the target genes.
*Functional evaluation by cell test
We can also provide evaluation systems that use higher cellular functions as indicators.
For application or inquiry, contact us at
To request an analysis, please specify the form of the sample (powder, solution, or other), the number of samples, and the nuclear receptor of interest.
The person in charge of analysis will call you back and explain the appropriate experimental design.