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Reporter genes can be divided into two groups: exogenous and endogenous reporter genes. Exogenous reporter genes are genes that are expressed in cells such as bacteria and are not found in mammalian cells; they are particularly suited for in vitro studies rather than in vivo as they tend to elicit an immune response inside living cells. However, because they are not found inside mammalian cells, they often have a higher specificity to their target of interest. Endogenous reporter genes on the other hand can be found inside mammalian cells and are often required to have a higher specificity to their target to distinguish them from similar proteins. One particular challenge is to introduce the reporter inside a living cell and be able to differentiate the expression and distribution of the gene in vivo. This is called non-invasive imaging and it has dedicated to it a whole field of research to discover novel ways to image inside a living cell.
Figure 1. Different reporter gene systems. Reporter genes are introduced into their target cell, either by vectors (in vivo) or fusion (in vitro). After reporter gene transcription, their products are various such as an antibody, a receptor, a flurorescent protein, an enzyme or a transporter. Imaging probes can be administered to in vivo applications which concentrates at the site of reporter gene expression. In vitro applications often use assays that are phenotypically characterised such as microscopy (Chuang & Cheng, 2010).
Exogenous Reporter Genes
Although there are many different types of exogenous reporter genes, some particular ones to mention include chloramphenicol acetyltransferase (CAT) which is a bacterial enzyme that detoxifies chloramphenicol by catalysing the transfer of an acetyl group from acetyl coenzyme A to the 3-hydroxyl position of chloramphenicol. A traditional CAT assay involves monitoring radioactive chloramphenicol and using thin layer chromatography (TLC) to separate the acetylated forms. The acetylated forms can be detected by having a higher migration rate than the unacetylated form of chloramphenicol, hence the amount can be determined by autoradiography or scintillation. An automated ELISA is also available to determine the levels of CAT without the use of radioisotopes making the assay much more manageable and it has been used in various assays such as testing for transfection efficiencies. However, the CAT assay is very limited it in its applications compared to other reporters in that it has a linear range and sensitivity.
Î²-galactosidase is another notoriously used bacterial enzyme that is widely used to monitor transfection efficiency using the blue-white screen. It is a hydrolase enzyme that catalyses the hydrolysis of Î²-galactosidase into monosaccharides. Using Î²-galactosidase as a reporter in the blue-white screen involves cloning the gene of interest into a lacZ promoter contained on a plasmid genome, thus interrupting the gene (Fig. 2). The cells are typically grown in the presence of X-gal, a chromogenic substrate and an inducer of Î²-galactosidase production, usually IPTG. If the gene of interest has successfully been ligated into a plasmid, colonies would appear white whereas cells that have a functional lacZ gene appear blue. Although there is some endogenous activity in mammalian cells, this can be reduced at higher pH levels.
Figure 2. Principle of the blue-white screen using Î²-galactosidase as a reporter gene, for the detection of recombinant vectors (Wilson & Walker, 2005)
Green fluorescent protein (GFP) on the other hand, is one of the most revolutionary reporter proteins in that it is vastly used and incredibly versatile and there are now many more colours available such as red and yellow. It was first discovered and isolated from the jellyfish Aequorea victoria and it was found that they naturally fluoresce without the need for other substrates or enzymes (*Shimomura, 2005). They are stable chromophore molecules produced by autocatalytic cyclisation without the need for a co-factor. GFP fluoresces in the lower green section of the visible spectrum with an emission peak of 509nm whereas Red Fluorescent Protein (RFP) or DsRed, discovered in Discosoma striata, has an emission peak of 583nm. As each fluorescent protein has a unique emission between 450nm and 650nm, one of the biggest advantages is that it is possible to image several targets of interest at the same time. GFP reporter imaging is often used in small animal imaging, however, low background to signal ratios and auto-fluorescence is a problem for this reporter gene as this limits sensitivity and specificity of the imaging. Fluorescent proteins can also create an immune response inside the host, again limiting their application in immunocompetent animals.
Similarly to GFP, luciferase is a family of enzymes that are able to catalyse the oxidation of a variety of substrates causing the release of light. This light emission can be detected by various apparatus such as a luminometer or a modified optical microscope. The most common luciferase enzymes are the firefly luciferase (Photinus pyralis) and the Renilla luciferase (Renilla reniformis, a sea pansy). The energy released as light comes from an energy dependent reaction involving the catalysis of luciferin and coelenterazine respectively whereby luciferin requires ATP but coelenterazine does not (*Iyer et al., 2005). Both have different emission spectra, with the firefly peaking at 490nm-620nm and Renilla at 480nm. They are both, however, immunogenic.
However, the most commonly used reporter gene is HSV1-tk or herpes simplex virus type I thymidine kinase and for this reason it is termed the gold standard reporter gene. HSV1-tk is similar to mammalian thymidine kinase type 1 (TK1) in that they are enzymes that converts thymidine to its phosphorylated form. However, HSV1-tk is less specific than its mammalian counterpart and so can phosphorylate a wide variety of different substrates whereas TK1 cannot. This difference has enabled the development of radiolabelled probes that are selectively phosphorylated by HSV1-tk. HSV1-tk can also be used as a therapeutic gene against viral infected diseases and tumours. Acyloguanosines are compounds that can be phosphorylated by HSV1-tk and once phosphorylated, they are able to kill cells by either blocking DNA synthesis or causing chain termination. Ganciclovir is one example of a pro-drug used for therapeutic purposes (*Johnson et al., 2001). However, despite its efficiency as a reporter gene and therapeutic gene, it produces a strong immune response when used. To overcome this problem, human mitochondrial thymidine kinase type 2 (hTK2) was developed and has a similar spectrum of substrate specificity to HSV1-tk (*Ponomarev et al., 2007).
Endogenous Reporter Genes
We now move onto the various types of endogenous reporter genes such as the dopamine 2 receptor (D2R). A cell surface receptor, it is most abundantly found in the brain striatum and pituitary gland and thus should not cause an immune reaction to this reporter gene. However, because they are found expressed primarily in these regions, the specificity of the reporter gene is limited. It often used as a reporter gene in nuclear medicine imaging by coupling the gene with radiolabelled probes like [11C]raclopride and 3-(2'-[18F] fluoroethyl)-spiperone (*MacLaren et al., 1999). However, targeting the D2R reporter can induce the activation of a G-protein coupled response resulting in decreased intracellular cAMP levels. To avoid this, a second generation D2R reporter, D2R80A has been developed (*Liang et al., 2001).
Another endogenous receptor protein is the transferrin receptor (TfR) which is a membrane receptor that transports superparamagnetic iron oxide particles into cells which can be visualised by MR (magnetic resonance) imaging. It is used in the imaging of gene expression and cell delivery by using human TfR lacking in the iron regulatory region and messenger RNA destabilisation motifs resulting in reduced feedback downregulation of receptor expression in response to iron uptake. TfR can also be expressed in tumours and may be used to enhance tumour detection and imaging if overexpressed. As TfR is expressed in vivo, this can severely limit its specificity. Also using MR imaging can be limiting as MR imaging sensitivity is low and so the concentration of the probe required is very high compared to the concentration required for PET imaging.
Î’-Glucuronidase (Î²G) is also noteworthy as it is an enzyme that is most commonly used to convert pro-drugs. It is selectively expressed in tumours and can be used to convert non-toxic prodrugs to active cytotoxic agents. Î²G can also specifically hydrolyse a non-fluorescent probe to one that is highly fluorescent for optical imaging. It is able to retain its imaging specificity whilst displaying low immunogenicity. However, glucuronide-based probes can be transported into bile through multiple resistance-associated protein 2 and subsequently become activated by the intestinal flora creating a non-specific signal from bacterial Î²G. There are many ways in which this problem is overcome (Chuang & Cheng, 2010), for example, mice can be pre-treated with antibiotics to clear intestinal microbes although this could lead to repopulation of pathogenic flora. Another method is to shunt the excretion pathway from the biliary to the urinary pathway. This is achieved by repeated administration of acetaminophen which will increase expression of multiple resistance associated protein 3 and drive the elimination of xenobiotic glucuronides through the urinary pathway. Probenecid can also inhibit multiple resistance associated protein 2 and reduce biliary excretion of glucuronides. Increasing urinary elimination may reduce the radioactivity retainined in the intestines. Another way is to replace the probe with one that exhibits predominant urinary excretion.
Antibody reporters are also attractive for research as they lend to the development of highly specific, non-immunogenic reporter genes. They are designed to elicit little to no immune response by using antibodies derived from the species of interest and humanised antibodies are able to minimise humoral and cellular immune response, allowing repeated and persistent imaging of gene expression in humans. Antibody-antigen pairs also possess high specificity and affinity without interference from cellular factors. The first antibody reporter developed was by Northrop et al., who produced an imaging probe consisting of fluorescein isothiocyanate coupled to the chelator diethylene triamine pentaacetic acid labelled with isotope. However, fluoroscein when incorporated into the chromosome can cause cell death or tumorigenicity. More recently, a membrane anchored anti-polyethylene glycol (anti-PEG) antibody has been developed that can trap a wide range of pegylated imaging probes. The anti-PEG reporter is often compared to the gold standard reporter gene, but does not however induce a humoral immune response. It has many advantages such as high affinity and specificity; it does not induce an immune response allowing continuous imaging and PEG is a water-soluble, non-toxic and biocompatible polymer that has been approved by the FDA. There are now many PEGylated probes such as pegylated superparamagnetic iron oxide, pegylated fluorescent probes and pegylated chelating agents (Chuang & Cheng, 2010). PEGylation therefore, may have the potential to be the next greatest reporter gene probe.
Applications of Reporter Gene Technology
Reporter gene technology is considered to be one of the most versatile experimental techniques available and can be applied to many different investigations. One such way is promoter analysis. This was probably one of the first applications of reporter gene technology whereby it was used to analyse the activity of promoters and/or enhancers or other genetic elements such as transcription factors. Specific expression of tissue receptors such as Î²1-adrenergic (#Bahouth et al., 1997) and interleukin-2 receptor (#Bamberger et al., 1997)genes, as well as genes that are targets for human disease, for example mammaglobulin (#Watson et al., 1998), a gene associated with breast cancer and transcription factors responsible for regulating gene expression have been identified using this technique. Similar to promoter analysis, gene expression often uses reporter genes to monitor the localisation of proteins and the pattern of expression using reporters such as Î²-galactosidase, CAT, luciferase and GFP.
Vectors expressing both the reporter and gene of interest and their subsequent transformation into cells, not only monitors gene expression but also acts as a way of screening for successfully transformed cells. This can be detected by using GFP as a marker and fluorescence activated cell sorting (FACS analysis). Transgenic animals, such as mice expressing GFP have been developed to monitor gene expression and their effects and are more commonly associated with identifying promoters and their regulation (#Chiocchetti et al., 1997). Other gene delivery systems involving reporter genes have in the past included reporters such as Î’-galactosidase and Î’-glucuronidase to monitor neural grafts (#Quintana et al., 1998) and transformation by agrobacterium in the plant, Brassica carinata (#Babic et al., 1998) respectively.
Imaging of gene expression has typically used fluorimetric and colorimetric assays using Î²-glucuronidase and Î²-galactosidase (Naylor, 1999), but these techniques have largely been overtaken by the development of GFP and luciferase systems. The advantages to using GFP and luciferase are that they give temporal and spatial information about a particular gene product, allowing us to look at the localisation of the product in the cell, protein-protein interactions and trafficking of the gene product around the cell. With this development of GFP and luciferase assays, multiple proteins can now be monitored at one time and also their response to changes in their surroundings can also be monitored in real time.
One major application of reporter genes is its use in drug discovery. They can be exploited to characterise receptors and ligands, for example, to identify agonist and antagonist ligands which alter receptor activity and one major receptor that has been characterised using reporters are GPCRs (G-Protein Coupled Receptors). Here, cell lines expressing a reporter gene and the receptor genes have been developed and are significantly involved in pharmacologically characterising these receptors. Screening for potential drugs by high throughput sequencing (HTS) using cell based assays is also a major use for reporter gene technology. Many different cell lines have been developed that express proteins of interest and by using HTS, these cell lines can be quickly screened due to the reporter genes acting as a marker and also as a quantitative marker, as they are able to measure the activation of specific signals from a transduction pathway.
Identifying signalling pathways is also a major application of reporter gene technology as it involves the elucidation of many different factors such as gene transcription, cell regulation and signal transduction proteins. These reporter gene assays play an important role in understanding the molecular basis of disease and also in finding new drug targets.
One recent example of reporter gene technology was used in a study by Beaulieu et al., (2010) that used fluorescence microscopy to screen for mutant genes in Mycobacterium tuberculosis (Mtb). Mtb is highly immunogenic in the host, eliciting a strong immune response from B cells and T cells and yet, our immune system often fails to fully eradicate Mtb. An estimated one third of the world's population is latently infected with this pathogen and 5-10% will develop active tuberculosis. This seemingly demonstrates Mtb's ability to elicit an immune response that is too weak to be cleared by the host, yet strong enough to cause damage in the lungs of a fully infected person with Mtb. What this paper questions then is "How does Mtb sculpt the host immune response to be quantitatively large but often qualitatively inadequate?" To begin to answer this question, they set out to gain a better understanding of Mtb's ability to modulate immunity.
It is thought that early innate immune responses to Mtb infection, affects the way in which our adaptive immunity responds to Mtb and one particular innate immunity component, macrophages, are found to be important for controlling bacterial growth but also in influencing other immune responses such as T- and B-cells. Recent studies have found the importance of macrophages in modulating the host-pathogen interaction (¥Kumar et al., 2010) and therefore, this study focused on the early encounter of Mtb with macrophages. To do this, they screened a large library of 10,100 Mtb mutants, to identify mutations that would lead to the dysregulation of the macrophage immune response. These mutants were generated by transposon insertion mutagenesis and due to health and safety constraints, limited to 12 host genes that responded to macrophages. The genes' expression could be detected microscopically using fluorescent proteins as reporters and were chosen as they were found to be transcriptionally regulated in mouse macrophage response to Mtb and they also represented a diverse range of molecules that are known to be involved in the immune response to infection, such as cytokines, chemokines, enzymes and cell surface co-stimulatory proteins.
Firstly, murine macrophage like RAW 264.7 cells were transfected with promoter-reporter constructs whereby the expression of three fluorescent proteins (AmCyan, DsRed, YFP) was driven by a promoter of a host gene and each of the twelve macrophage cell lines underwent clonal selection depending on their fluorescence activity. For example, unstimulated cells were sorted by flow cytometry to isolate populations with minimal resting fluorescence and further sorted by separating out cells with high levels of expression when stimulated with IFNÎ³ and these cells were expanded as individual clones; fluorescence microscopy was also used to confirm the induction of fluorescence. In the primary screen, assay plates containing one clone with a given promoter via one of the three fluorescent reporter proteins were each infected with an Mtb mutant and alterations in fluorescence induction were monitored by automated microscopy. A secondary screen followed whereby mutants selected from the primary screen by their ability to dysregulate fluorescence, underwent further experiments to confirm their activity of dysregulation of reporter induction. These screens identified 364 candidate immunoregulatory genes which were further investigated via qRT-PCR (quantitative Real Time-PCR) to verify their impact on Mtb's immune expression to primary macrophages. Focus was placed a specific mutation in Mtb, whereby transposon mutagenesis had disrupted a gene encoding a conserved protein of unknown function. This mutant elicited a stronger immune response in vitro than wild type Mtb and was attenuated in the mouse. What Beaulieu et al., have essentially achieved is the revelation of a catalogue of mutant strains, for further exploration into how Mtb modulates immunity and potentially promote these immunoregulatory mutations into research for a potential vaccine against Mtb.
As we can see, this paper clearly shows the significance of reporter gene technology and how useful it is in research, as for instance, the use of various different fluorescent proteins in the screens, demonstrate s the versatility of reporter genes to identify the transcription of several immune compounds such as cytokines and chemokines at the same time. Problems with reporters can arise however; for example due to the size of the mutant screens, there were many technical challenges involved such as maintaining reporter gene sensitivity in the macrophages and their response to changes in the bacterial innoculum. This was particularly difficult and was illustrated in the fact that a large false positive rate was seen in the primary screen. Nevertheless, a high percentage of the mutants were confirmed in the secondary screen, again by fluorescence microscopy, to dysregulate the immune response of primary macrophages. Despite the problems involved with reporter gene technology, the benefits are vast and numerous in number and they are hugely adaptable to almost any challenge that will be faced in science and with the constant development of new reporter gene assays, the technology is sure to become synonymous with many different research aspects.