Or when luciferase is positive because of a coincidence
Sometimes an experiment is working but our interpretation of results is not. The underlying assumption of any reporter gene system is the relationship between the assay readout and the molecular/cellular mechanism for which the assay has been engineered to report.
For instance, from a PPAR-responsive promoter placed upstream the luciferase gene we will expect to observe more light emission when PPARs nuclear receptors are active, with the amount of light generated that is somewhat proportional to the degree of PPAR activation. Thus, if you transfect a cell line with a PPRE-luciferase plasmid, and treat the cells with a drug supposed to be a PPAR agonist, you would expect an increase in luciferase readings. Imagine this is the case when studying a new molecule not previously known to modulate PPAR actions. You maybe excited. I assume you already compared the effect of your molecule with parallel cell preparations that were not treated. I also assume that you already controlled each measurement with an internal reference like a Renilla reniformis plasmid, so that you can normalize for differences in cell number due to handling procedures or to the molecule itself. And I further assume you already tested the vehicle used to put the molecule in solution: sometimes dmso or ethanol may influence cell lines. Good, maybe you have found a new PPAR ligand. Here is the point when you should absolutely read a recent Nature Methods letter signed Cheng and Inglese. Most likely, you will not read the paper and, perhaps urged from your excited supervisor, you will start asking which PPAR is targeted by your molecule: turns out there are at least three PPAR isotypes. Thus, you may want to check the drug effect in the same cell line in which each of the three PPAR isotypes have been previously knocked down: weeks of set up. Or you may prefer tailoring the luciferase assay so that it become responsive to a single PPAR, for instance using the gal4-hybrid approach: weeks of cloning. In the while, the boss will start looking around for PPAR KO mice. However, your following results start to be less clear, and you already performed some binding assays that were inconclusive. Please read the Cheng and Inglese paper! No, you think, either your drug is not a PPAR ligand but indirectly modulate a cellular pathway responsible for the final PPAR activation, or the drug is actually a ligand, but requires a third factor like an ancillary protein or a PPAR post-translational modification that is not present when using the recombinant protein in the binding assay. You are at a point in which you trust the luciferase results but do not progress with the story. You maybe stalling for months. It's frustrating. And when you are frustrated, you turn your thoughts in directions other than the frustrating source, so you notice the Nature Method print magazine provided free to you thanks to Reportergene's complimentary subscription. Got it? Open the October issue, page 937 and read the paper I was mentioning: more than 80% of the apparently active compounds are assay artifacts. Have you ever thought that your molecule is a luciferase agonist and not a PPAR agonist?
To distinguish compounds that target a biological pathway from those that interfere with a reporter, we designed a coincidence 'biocircuit' based on the principle that it is easier to tell signal from noise when the signal is reported by two or more detectors. This concept is implemented here using co-translational expression of nonhomologous reporters: for example, proteins having different catalytic, light-emitting or fluorescence properties.
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Ken C-C Cheng & James Inglese. A coincidence reporter-gene system for high-throughput screening - Nature Methods, October 2012 doi:10.1038/nmeth.2170