Researchers interested in using genetic reporters to study gene expression should consider using the Promega Luciferase Assay System. This popular kit is relatively quick, easy to use and involves little hands-on time. In general, firefly luciferase is extensively used as a reporter because one only needs a few seconds to assay each sample. The mechanisms of the assay are quite simple—light is produced from an enzymatic chemical reaction, which can then be measured by the scientist with the use of a luminometer. More specifically, the light comes from the oxidation of luciferin to oxyluciferin by the luciferase enzyme. However, in much of the older conventional assays, the light generated quickly decays. The Promega Luciferase Assay System overcomes this obstacle by including coenzyme A (CoA) with the provided luciferin substrate, allowing for greater enzymatic turnover that increases light intensity and maintains light emission for about one minute.
To get started, one needs to subclone their favorite DNA regulatory region into a luciferase reporter vector that can then be transfected into a variety of cell types. Cell lysates, obtained with a lysis reagent provided with the kit, can then be directly assayed for levels of the expressed luciferase protein. All one needs to do is add an amount of cell lysate to the luciferase assay reagent and measure the intensity of emitted light by using a luminometer.
Like many researchers studying gene expression and cellular events coupled to gene expression, I have used the Promega assay system frequently. Some of my work involves the study of gene activation by specific transcription factors. To determine whether activated transcription factors play a role in particular signaling pathway, I transfect a luciferase construct bearing the promoter elements that bind to these complexes into cells. If my favorite transcription factors are indeed activated in these signaling pathways, they would then bind to and induce transcription of the luciferase gene. Depending on what the experimental variables are, I would compare the relative values of light units from my samples to gauge the involvement of my favorite transcription factors in gene expression. As a result, I can (and often do) get quick answers to many of my initial questions.
This system is extremely sensitive, a fact that can sometimes be a drawback. Because it is so sensitive, one can encounter readings with high background (i.e., a high basal reading that may skew one’s ability to interpret actual cellular events). This may actually depend on which luciferase vectors are used, and also on which regulatory elements/signal transduction pathways are being studied. Also, it is important to keep in mind that results obtained from reporter gene assays do not necessarily reflect actual physiologic events.
Nevertheless, a significant positive aspect of the Promega assay system is that it is easy to use. Because the luciferase protein does not require post-translational modification, one can start the assay immediately after obtaining cell lysates. This is especially important when one needs to quickly acquire information about cellular events involving their favorite proteins and signaling pathways.
Joe Lau
Graduate student
Mount Sinai School of Medicine