Gene reporter assays have contributed hugely to our understanding of how genes are regulated during growth and development, for example, through the study of spatiotemporal gene expression patterns, as well as how gene expression is regulated by transcription factors, gene regulatory elements (so-called cis-acting elements), and exogenous regulators (trans-acting factors). Besides investigating gene regulation, gene reporter assays are also useful in transfection experiments, both to optimize and standardize transfection efficiencies and to screen transfected cells in routine workflows.
What Happens in a Reporter Gene Assay?
In any reporter gene assay the activity of a reporter gene is measured under a given set of conditions, for example, over a time course, in the presence of drugs, or under varied environmental conditions.
A typical reporter gene assay setup is as follows:
- A reporter gene is fused with a target regulatory DNA sequence in an appropriate expression vector.
- The recombinant vector is then transfected into the cell type of choice.
- The reporter gene is transcribed and translated by the transfected cells, and depending on the actual reporter gene used, its activity may be detected directly or indirectly through the ability of its encoded protein to convert a substrate to a detectable product. The latter scenario is illustrated in Figure 1.
Figure 1. Illustration of typical reporter gene assay. Reporter gene is fused to the promoter of the gene of interest. When the promoter is on, the reporter gene is transcribed and translated into an enzyme that reacts with a substrate to product a detectable product.
What Makes For a Good Reporter Gene?
- It should encode a protein whose activity can be easily detected with high sensitivity over any endogenous gene activity. Ideally, the reporter gene is not expressed endogenously at all.
- The signal generated by the reporter gene product should have a broad linear detection range for maximum utility and quantitative results. The assay should produce reproducible results.
- The presence and expression of the reporter gene should not affect the general health and physiology of the transfected cells.
What Can You Do with Reporter Gene Assays?
As alluded to above, reporter gene assays have revolutionized the way we study gene regulation, and the broad choice of assay formats available means that these assays can be used in diverse species, encompassing plants, animals and microbes. Let’s look at a few common situations where a reporter gene assay might advance or simplify your research.
Functional Characterization of a Transcription Factor
Transcription factors are trans-acting regulatory factors which bind specifically to enhancer regions of DNA upon receiving an appropriate cellular signal, which ultimately results in an increase or decrease in the expression of certain target genes. Transcription factors play pivotal roles in signal transduction processes, but they exert multiple downstream effects and thus may be difficult to study without some kind of easily measurable output signal.
Without a reporter system to study a transcription factor, one would need to go through the cumbersome process of isolating cellular RNA, performing reverse transcription (RT) to cDNA and running RT-PCR to monitor target gene expression every time an experimental condition was altered. The use of reporter gene assays greatly speeds up this workflow since one can simply clone the reporter gene downstream of the regulatory DNA sequence in a vector, and then transfect this vector into the cell type in which the target transcription factor is under investigation.
The advantage of using a reporter assay to study a transcription factor is that you quantify the activity of the protein that results from target gene expression, which may be more biologically relevant than studying target gene mRNA levels. However, reporter assays are not suitable if information about mRNA turnover is desired. As a side note, one can also use reporter assays to simply study the expression patterns of gene(s) of interest, without necessarily considering the involvement of particular transcription factors.
Characterization of Gene Promoters and Enhancers
Here, a reporter gene is cloned either downstream of a promoter or an enhancer under investigation, and the resulting construct is transfected into cells. High or low expression of the reporter gene(s) indicates that the promoter or enhancer under investigation is strong or weak, respectively. Reporter assays that investigate the strength of promoters and enhancers may be used as part of a larger workflow to understand complex gene regulatory networks.
Screening for Transfected Cells and Measuring Transfection Efficiency
A transfection assay to screen for or detect transfected cells may be used as an alternative to traditional selectable markers. While selectable markers, which are most often antibiotic resistance genes, allow transfected cells to grow in the presence of some exogenously applied agent and can thus be used to identify transfected cells, they lack quantitative value.
To measure the transfection efficiency of any plasmid into (usually) eukaryotic cells, a reporter gene e.g., green fluorescent protein, is cloned downstream of a constitutive promoter in a plasmid, and the resulting construct is then co-transfected along with the experimental plasmid. Expression of the reporter protein can be detected in several ways, most commonly by flow cytometry, and its quantification may be used as a measure of transfection efficiency.
Many Reporter Gene Assays To Choose From
Fortunately, there are several reporter gene assay setups to choose from. Which one is right for you will depend on your experimental goal i.e. are you studying gene regulation or do you just want to measure transfection efficiency?, as well as the organism and cell type, type of result sought (spatial or temporal), and the preferred detection method (histochemical staining, scintillation counters, spectrometry, fluorimetry or luminometry).
The table below provides an overview of the most widely used reporter gene assays.
|Green fluorescent protein (GFP)
|Cells that express GFP glow fluorescent green under a UV light.
Yellow (YFP) and red (RFP) versions are also available, allowing the investigation of multiple genes at once.
|Commonly used to measure gene expression and study gene regulation.
Note: A specialized microscope is required to see individual cells, while individual cells can be counted by flow cytometry (this is often used to monitor transfection efficiency).
The encoding gene is most often from the firefly species Photinus pyralis, although genes from other firefly species may also be used
|The luciferase enzyme reacts with a substrate (usually luciferin) to produce yellow-green or blue light, depending on the luciferase gene. Since light excitation is not needed for luciferase bioluminescence, auto fluorescence is minimal and the fluorescence signal detected is virtually background-free.
This assay may be more time-consuming than the GFP assay, because one needs to collect the transfected cells, lyse them to release the proteins, add luciferin, and then measure the luciferase activity using a luminometer. Despite this fact, luciferase assays are often favored over GPF assays because of their extreme sensitivity, allowing one to detect even minute changes in gene expression.
|Widely used to measure gene expression and study gene regulation.
Species-dependent differences in light emission from luciferase enzymes make it possible to set up multiplex assays, using multiple luciferase reporters simultaneously to study several gene regulatory elements at once.
|Several assay formats available. GUS staining, a histochemical method, is the most widely used. Here, the GUS gene is fused with the promoter or regulatory element of interest. When expressed in the presence of certain exogenously added colorless or non-fluorescent substrates, GUS converts them into colored or fluorescent products that can be readily detected.
The GUS assay offers great flexibility with a choice of different GUS substrates available, suitable for different modes of detection – histochemical, spectrophotometric or fluorimetric. Histochemical detection provides an excellent method for analyzing single cells.
The drawback of the GUS assay is that the cells are killed in the process.
|It is often used to study gene regulation in plants.
|lacZ (blue-white screen)
|The bacterial lacZ gene encodes a β-galactosidase enzyme. Active β-galactosidase is readily detectable upon the addition of certain chromogenic substrates, the most widely used is known as X-gal. β-galactosidase reacts with X-gal to produce an intense blue product that is easy to identify with the naked eye, and may be quantified spectrophotometrically.
|Widely used in cloning to identify transformed bacteria harboring recombinant plasmids. Insertion of ‘foreign DNA’ into the multiple cloning site of an expression plasmid interrupts the lacZ gene, thus inhibiting β-galactosidase production. Recombinant bacterial colonies therefore appear white while non-recombinant colonies appear blue.
Over to You
Do you use reporter gene assays for a purpose that we haven’t covered here? Or maybe you are using completely different reporter genes to the ones we mention? We would love to hear from you, so do drop us an email and share your experiences with us!
Stay tuned for our next article where we will look at fluorescent reporters with functionalities!
Article by Karen O’Hanlon Cohrt PhD. Contact Karen at firstname.lastname@example.org.
Karen O’Hanlon Cohrt is a Science Writer with a PhD in biotechnology from Maynooth University, Ireland (2011). After her PhD, Karen moved to Denmark and held postdoctoral positions in mycology and later in human cell cycle regulation, before moving to the world of drug discovery. Her broad research background provides the technical know-how to support scientists in diverse areas, and this in combination with her passion for writing helps her to keep abreast of exciting research developments as they unfold. Follow Karen on Twitter @KarenOHCohrt. Karen has been a science writer since 2014; you can find her other work on her portfolio.