by Catherine Shaffer
Reporter gene assays are used in a variety of ways in biological research, from in vivo and in vitro screening to gene expression and cell signaling. In essence, a reporter gene is simply a measurable marker within the experimental system, often a replacement for a value that would be difficult to measure otherwise. The gene is cloned into cells with a DNA sequence of interest, and the positive signal in the reporter assay indicates that the target DNA is also present.
Assays using chemiluminescence
Common methods of reporter gene detection include firefly luciferase, beta lactamase, and green fluorescent protein. Of these, luciferase is the most widely used method. It has become a standard for quantitative reporting in applications like gene regulation and signal tranduction. Firefly luciferase is easy to use, quick, accurate, and sensitive. Promega is well known for commercializing luciferase as a reporter gene assay. Says Kevin Kopish, global product manager for Promega, “What made luciferase so popular was that it really simplified the process of doing reporter gene assays. Previously, you had to wait long periods of time to get your results. Luciferase gave you all the sensitivity of any other technology while also being easy to use.”
These advantages are integrated into Promega's pmirGLO dual luciferase miRNA target expression vector. Dual reporter assays are popular because they simultaneously measure two reporter genes in a single system, and normalize the signal from the experimental reporter to a control reporter. The pmirGLO inserts miRNA target sites in the 3' UTR of the firefly luciferase gene (luc2). This results in a reporter signal when endogenous or introduced miRNAs bind to the target sequence.
Gaussia luciferase
Novel luciferases from other organisms have become available for use in gene reporter assays. The most notable of these are the Gaussia luciferase, from Gaussia princeps; two Luciola luciferases, one red-emitting and one green-emitting, both from the firefly Luciola italica; and Cypridina luciferase from Vargula Hilgendorfi. Targeting Systems developed and commercialized these luciferases as reporter genes (expressed in a plasmid or lentivirus) for high throughput screening (HTS) and other applications.
The increased brightness of these luciferases is what allows for their use in HTS and other new applications. Gaussia luciferase is 1000 times brighter, red Luciola is 100 times brighter, and Vargula 50 times brighter than the traditional firefly lucifarases. The increased signal intensity offers tremendous advantages in drug discovery applications (HTS) and in photodynamic therapy (PDT) which uses luciferases in combination with photosensitized dyes for selective killing of target (e.g. cancer) cells. The red Luciola luciferase has proven to be particularly advantageous for deep tissue imaging due to its intense brightness; it also offers reduced background due to lower absorption of red light by tissue. Targeting Systems offers 4 additional luciferase reporter systems for enabling multiplexed luciferase reporter assays. The most notable is a secreted dual luciferase assay (using the Gaussia and Vargula reporters) and several triple luciferase assays that use a single assay reagent. In addition, two novel green and violet emitting luciferases are scheduled for release in the near future.
Rampyari Walia, PhD, CEO of Targeting Systems says, “We have worked hard to develop each of these luciferases as commercially viable assay systems. The Luciola luciferases we offer are 1000-fold brighter versions of the traditional enzymes. A wide spectrum of luciferases will now enable analysis with 4 different luciferase reporters within the same cell, thus saving screening time.”
Simplicity and convenience in a colorimetric assay
Another useful reporter system is secreted alkaline phosphatase (SEAP). SEAP differs from endogenous alkaline phosphatase in that it is heat stable and not inhibited by L-homoarginine. As a reporter, SEAP offers the advantage of monitoring gene activity without lysing the cells. SEAP catalyzes the hydrolysis of pNitrophenyl phosphate (pNpp), yielding a colorimetric readout at 405 nm. InvivoGen offers a wide range of assays compatible with the SEAP colorimetric reporter system, including a large product line dedicated to the innate immune system. Two other product families, QUANTI-Blue™ and HEK-Blue™ Detection media, use the similar chemistry to the basic SEAP assay, except that the substrate changes to a blue color. Says Scott Vara, sales and support manager for InvivoGen, “We really like SEAP because of the fact that it's quantifiable and secreted. With luciferase you have to do a cell extract. SEAP is very simple, very convenient.”
Green fluorescent protein
One of the disadvantages of a reporter system based on a secreted molecule is that it can't help with isolation of cells expressing the gene of interest. Creating chimeric proteins using green fluorescent protein is a reporter method that can be used when it is necessary for the signal to remain in the cell. This type of assay has proven to be very useful for immediate, non-invasive visualization and monitoring of the differentiation status of stem cell populations. The MilliTrace GFP reporter cell lines from Millipore are designed for embryonic and neural stem cell research, in human and rodent models. Constitutively expressing and lineage restricted reporter stem cells, including nanog and nestin GFP reporter cells, are available within this range of products. Says Louise Rollins, product manager for Millipore Corporation, “In the past, the only option for many researchers has been to create their own reporter cell lines using separately purchased components, which can be inconvenient, time-consuming, and costly. The MilliTrace GFP Reporter Cell Lines are ready-to-use, and provided with optimized media for maximum convenience.”
Constructing reporter cell lines can be an arduous process of six to nine months. A novel product from Affymetrix, the QuantiGene® ViewRNA Assay, is a cell-based mRNA in situ hybridization assay that averts the need for labor-intensive reporter cell lines. The QuantiGene Assay allows researchers to measure single RNA molecules in individual cells in a multiplex format, quantifying endogenous expression of the natural gene. The assay, which is based on patented branch DNA signal amplification methodology, can quantify expressed RNA transcripts by locating and counting individual RNA molecules in individual cells, or by measuring the total light emission of the expressed gene of interest in a population of cells in a microwell.
The new assay has broad applicability in research and industry. It can be used as an alternative to high-throughput phenotypic or reporter gene screening applications in HTS primary and secondary compound screening. Other applications include biomarker validation, testing cells or FFPE tissues for gene expression as an alternative to immunohistochemistry, in vitro or in vivo RNAi delivery, siRNA knockdown measurements and transcriptional heterogeneity studies. Says Kim Crawford, director of marketing for QuantiGene products, “Reporter genes have played a major role in studying a wide range of biological areas, from gene expression and control to cell signaling and drug discovery...However, they still have limitations in that genetic engineering is employed, altering the phenotype of the cell and/or trafficking of the native protein that has been tagged. Stable cell line expression is time consuming and clone selection of best expressers can drift over time.” One limitation of a QuantiGene type of assay is that it is not suitable for real-time monitoring of transcript levels, because the cells are processed and fixed.
Gene reporter assays are increasingly used in studies such as high throughput screening, tagging and following proteins in real time, monitoring live cell cultures using secreted reporters or membrane-anchored secreted reporters, and in vivo imaging of deep tissues in animals. A number of reporter systems are available, including colorimetric, fluorescent, and chemiluminescent options. As well, new options are available for using reporter genes without the need to establish a stable cell line for expression.