|Courtesy of Applied Biosystems|
Reporter genes are generally joined to a regulatory DNA sequence in an expression vector that is usually propagated in Escherichia coli before transfection into the cell type of interest.1 A control reporter driven by a strong, constitutive promoter is cotransfected with the experimental reporter plasmid to normalize for transfection efficiency and to account for the fact that expression of the experimental reporter may vary in different cell types. After allowing time for gene expression, the cells are assayed for reporter mRNA, the reporter protein itself, or for the activity of the reporter protein. Detection of the reporter gene product usually requires cell lysis, although some products are amenable to in situ analysis.
Ideally, a reporter gene encodes for a protein whose activity can be detected with high sensitivity above any endogenous activity and that displays a wide dynamic range of response (over several orders of magnitude). The assay should be safe, easy to use, relatively inexpensive, and provide rapid and reproducible results.2 Choosing the best reporter gene assay depends on the type of study (regulation of gene expression or determination of transfection efficiency), organism and cell type, type of information sought (temporal versus spatial), and preferred detection method (e.g., histochemical staining, liquid scintillation, spectrophotometry, or luminometry) Many reporters have been adapted for a broad range of assays, including colorimetric, fluorescent, bioluminescent, chemiluminescent, ELISA, and/or in situ staining. This article discusses the most commonly used reporters and representative assay systems.
|Courtesy of Novagen Inc.|
|Courtesy of Novagen Inc.|
Amersham Pharmacia Biotech of Piscataway, N.J.'s Quan-T-CAT™ Assay System measures CAT activity with [3H]-acetyl-CoA and biotinylated chloramphenicol. Researchers capture the chloramphenicol using streptavidin-coated polystyrene beads, and measure the extent of acetylation using a scintillation counter. According to product manager Fiona Fitzgerald, this assay is about 30 times more sensitive than 14C-based assays. Researchers that want to avoid the expense and safety concerns associated with radioactive compounds can turn to Stratagene of La Jolla, Calif.'s Flash® CAT kit. This system uses BODIPY® (borondipyrromethane) difluoride -labeled chloramphenicol, whose activity is quantified using a fluorometer or fluorescence scanner.
b-galatosidase (b-gal): The E. coli enzyme b-gal (encoded by lacZ) hydrolyzes galactoside sugars such as lactose. Although the background levels of activity present in many bacteria, plants, and animals limit its application, b-gal is still frequently used as a control reporter. Using a special buffer at pH 8, like that included in Costa Mesa, Calif.-based ICN Biomedical's Aurora™ Gal-XE kit, helps to discriminate the E. coli b-gal from any endogenous enzymatic activities. There are several assay formats: colorimetric, fluorescent, and chemiluminescent.
Hydrolysis of o-nitrophenyl-b-D-galactoside (OPNG) or chlorophenol red b-D-galactoside (CPRG) yields colored products that can be quantified spectrophotometrically. In situ b-gal expression can be visualized histochemically through hydrolysis of X-Gal (5-bromo-4-chloro-3-indoyl-b-D-galactopyranoside), which yields a blue precipitate, or using fluorogenic substrates such as b-methyl umbelliferyl galactoside (MUG) and fluorescein digalactoside (FDG).
The FluoReporter® lacZ Flow Cytometry Kits from Molecular Probes, and the in vivo lacZ b-galactosidase Detection Kit from Marker Gene Technologies (MGT), both of Eugene, Ore., provide materials and protocols for quantifying b-gal activity in single cells using flow cytometry or with confocal microscopy with FDG. However, there is a drawback to using FDG as a substrate: FDG and its fluorescent hydrolysis product, fluorescein, rapidly leak from cells under physiological conditions.
To reduce this leakage, MGT's kit includes a protocol developed by Len Herzenberg and colleagues at Stanford University that incorporates a cooling step. Molecular Probes adopts an alternate approach with the Detectagene™ and Imagene™ kits. Chip Walker, product manager for life sciences at Molecular Probes, says, "Most kits use the very basic FDG substrate, but the Detectagene and Imagene kits use modified FDG substrates, which greatly improve cell retention." The substrates in DetectaGene Green and Blue lacZ Gene Expression Kits are galactose derivatives containing a mildly thiol-reactive chloromethyl group. These groups react with glutathione and possibly other intracellular thiols to form a peptide-fluorescent dye adduct that does not readily cross the plasma membrane and therefore is much better retained than the free dye. The ImaGene Green and ImaGene Red lacZ Gene Expression Kits use fluorescein- and resorufin-based galactosidase substrates, respectively, which contain a 12-carbon lipophilic moiety. Cleavage of these substrates produces fluorescent products that are retained by the cells, probably by incorporation of their lipophilic tails into cellular membranes.
|Courtesy of Molecular Devices|
Several companies now offer kits for the quantification of b-gal, employing chemiluminescent dioxetane chemistry that widens the dynamic range, increasing detection sensitivity over comparative colorimetric or fluorescent assays. The Galacto-Light™ and Galacto-Light Plus™ kits, from Applied Biosystems of Foster City, Calif., are ideal for studying weak promoters and can be completed in less than one hour. The Gal-Screen® System, also from Applied Biosystems, is amenable to high-throughput screening of mammalian or yeast cells in microwell cultures, because it incorporates the cell lysis components, dioxetane substrate, and enhancer in a single reagent with no need for automatic injection.
b-glucuronidase (GUS): Another E. coli-derived hydrolyzing enzyme that lends itself to a variety of assay formats, GUS hydrolyzes glucuronides and is most commonly used in studies of viral plant pathogens, plants, yeast, and fungi that lack endogenous activity. ICN's Aurora GUS includes bacterial GUS for use in plant and animal reporter gene assays, and dioxetane substrate, creating a kit that is more sensitive than equivalent fluorescent assays that incorporate 4-methylumbelliferyl-b-D-glucuronide (MUG). The assay is not affected by fluorogenic compounds in plant material or subject to disturbing quenching effects at high protein levels.
Secreted alkaline phosphatase (SEAP): A mutated form of human placental alkaline phosphatase (PLAP), SEAP is a reporter gene that offers the great advantage of being secreted outside the cell. Thus, its activity in the same cell sample can be measured nondestructively and repeatedly over time using an aliquot of the culture medium. This saves the time normally required to prepare cell extracts and allows cells to be studied further after assaying for SEAP. Also, the truncated form of human PLAP normally used in reporter gene assays is heat stable and exhibits resistance to L-homoarginine, permitting any endogenous AP activity present in a cell population to be eliminated by preheating and assaying in the presence of L-homoarginine.
Applied Biosystems' Phospha-Light™ kit uses a sensitive dioxetane substrate for AP, called CSPD®, which can detect ultra-low levels (about 10-21 moles) of the enzyme. Similarly, the chemiluminescent technology available in ICN's Aurora AP, "offers excellent sensitivity compared with other reporter systems, enabling detection at very low [levels] of expression," says Lauren Schultheiss, ICN's cell biology product manager. BD Biosciences-CLONTECH of Palo Alto, Calif.'s Great EscAPe™ Reporter System is available in either a fluorescent format, using 4-methylumbelliferyl phosphate (MUP), or in a chemiluminescent format, using CSPD.
Luciferase (luc): The bioluminescent enzyme luciferase, cloned from the North American firefly (Photinus pyralis), catalyzes the oxidative carboxylation of beetle luciferin, emitting photons that can be measured using a luminometer or scintillation counter. Luciferase-based assays have become widely used because they are rapid, convenient, and have a broad linear range over seven or eight orders of magnitude. In addition, since the enzyme is not post-translational modified, luciferase activity is closely coupled to protein synthesis. Finally, its great sensitivity (detection of less than 10-20 mol of luciferase) and short half-life make it ideal for assessing inducible effects in transient assays measuring temporal gene expression.3 However, in the past, detection of the flash response required a luminometer with an automatic injector to measure the peak light emission occurring within 0.3 seconds of substrate addition. Fortunately, adaptations of the chemical reaction produce a more stable signal by reducing feedback inhibition, allowing the use of scintillation counters or luminometers without injectors.
For example, the Wallac GeneLux kit from PerkinElmer Life Sciences of Boston stabilizes light emission with dithiothreitol, pyrophosphate, and bovine serum albumin, whereas the Luciferase Gene Assay, high sensitivity, from Roche Molecular Biochemicals of Indianapolis, stabilizes the luciferase signal for several minutes using CoA. Light emission with a half-life of about three hours is possible using Roche's Luciferase Assay with Constant Light Signal, which stabilizes the reaction using AMP, while the LucLite® and LucLite Plus, Constant-Quanta™ reporter gene assays from Packard BioScience of Meriden, Conn., offer a long-lived "glow"-type signal with a half-life up to five hours.
Madison, Wis.-based Promega Corp. and Applied Biosystems offer other improvements to the luciferase assay. Promega's Dual-Luciferase® Reporter Assay System combines the benefits of the firefly luciferase assay with an assay for sea pansy (Renilla reniformis) luciferase to provide a rapid, single-tube (or single-well), dual-reporter assay for internal normalization of gene expression measurements. The Renilla luciferase is structurally different from firefly luciferase and uses coelenterazine as a substrate, making possible the quenching of firefly luminescence and simultaneous activation of Renilla luciferase with the addition of Promega's Stop & Glo® Reagent.
Applied Biosystems' Tropix® Dual-Light® assay is used for the detection of luciferase and b-gal sequentially in a single sample. "The Dual-Light kit is the only dual reporter kit on the market for the detection of b-gal and luciferase with the b-gal reporter, providing an excellent robust control for the normalization of cell number," says Mark Roskey, marketing director for Applied Biosystems' Tropix products. These dual assays are simple, rapid, and convenient without compromising sensitivity or dynamic range.
Green fluorescent proteins (GFP): GFP, derived from the Pacific Northwest jellyfish (Aequorea victoria), fluoresces upon UV irradiation and is an ideal in vivo marker of gene expression because, unlike other reporters, it requires no substrates or cofactors to fluoresce and retains its activity in the presence of heat, denaturants, detergents, and most proteases. It is also easy to assay using fluorescence microscopy or fluorescence-activated cell sorting (FACS). Anthony Salerno, molecular biology product manager at Lincoln Park, N.J.-based CPG (www.cpg-biotech.com), explains, "The main advantage of fluorescent protein vectors is that you can look under a microscope at individual living cells and see where fluorescent fusion proteins are localized and watch such processes in a dynamic fashion." Because wild-type GFP is not as sensitive as luciferase, and since GFP can not be amplified enzymatically, many companies have engineered vectors containing mutants of GFP that are brighter, differentially colored, and localized to different cell compartments.
GFP is generally nontoxic as exemplified by the production of transgenic mice that glow green when GFP is integrated into their DNA. However, some users of Aequoria GFP have difficulties with unstable expression caused by cell toxicity. Stratagene has cloned a GFP cDNA from the sea pansy, R. reniformis, to create their new low-toxicity, humanized Renilla GFP, Vitality™ hrGFP.4 Stratagene's internal data has shown that high-level expression of functional fluorescent protein in retrovirus-transduced cells is more consistent and less toxic over time and passage number for hrGFP than for Aequoria GFP. Thus, cell lines transfected with the Vitality vectors exhibit more stable, long-term expression due to lower cell toxicity.
The AFP-Tag™ vectors from CPG are blue and green protein-tagging vectors that express the inserted gene of interest as an autofluorescent fusion protein. The red-shifted GFP and Blue Fluorescent Proteins (BFP) are GFP mutants that show increased specific autofluorescence at different excitation and emission spectra and, therefore, can be detected independently. Since there is no overlap in emission spectra of the blue and green fusion proteins, AFP-Tag vectors can be used individually or in dual-color labeling assays. These fusion proteins offer highly stable autofluorescence that can be visualized in either live or fixed cells and are available for bacterial expression and for both transient and stable transfection into eukaryotic cells.
Carlsbad, Calif.-based Qbiogene's SuperGlo™ GFP and SuperGlo BFP (www.qbiogene.com) demonstrate enhanced excitation peaks and improved emission spectra that make them particularly suited for monitoring protein interactions in living cells using FRET (fluorescence resonance energy transfer). Finally, BD Biosciences-CLONTECH features a fluorescent protein that changes color from green to red over time (see p. 24), thus signaling when the promoter of interest has become inactive (Living Colors™ Fluorescent Timer).
Stratagene's PathDetect trans-reporting systems allow researchers to determine if their gene of interest directly or indirectly activates the c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase (MAPK), cAMP-dependent kinase (PKA), or the p38 kinase pathways. The systems include a pathway-specific trans-activator plasmid to be cotransfected with a trans-reporter plasmid encoding either CAT, b-gal, SEAP, luciferase, or hrGFP.
|Courtesy of Thermo Labsystems|
Finally, San Diego-based Gene Therapy Systems (GTS) offers PNA-dependent gene chemistry, described as "a new and unique twist on reporter genes," by Kal Masri, director of licensing at GTS. PNAs (peptide nucleic acids) are oligonucleotide analogs that hybridize in a sequence-specific manner to complementary DNA targets (for example, on a plasmid), but which can be conjugated to a ligand, fluorophore, or antibody, allowing the DNA molecule itself to be followed. Thus, a gene of interest can be converted into a "reporting gene" by insertion into the multiple cloning site of the pGeneGrip™ vector and attachment of a PNA clamp labeled with rhodamine, fluorescein, biotin, or maleimide. Because these clamps are either labeled with a fluorophore or a functional peptide, both DNA distribution and functional protein/peptide studies can be easily performed. Vectors expressing GFP, b-gal, luciferase, or SEAP reporter genes and prelabeled with PNA clamps are also available.
Reporter gene technology keeps with the times by becoming useful in high-throughput drug screening, in monitoring gene therapy attempts, and in the construction of biosensors to detect contamination in environmental samples such as water and soil. A variety of well-characterized reporters are available and, recently, unique methods of reporting have shed "new light" for scientists. The investigator need only select which reporter is most suitable for the study at hand.
1. D. Groskreutz, E.T. Schenborn, "Reporter systems," Methods in Molecular Biology, 63:11-30, 1997.
2.J.C. Lewis et al., "Applications of reporter genes," Analytical Chemistry, 70:579A-585A, 1998.
3. L.H. Naylor, "Reporter gene technology: The future looks bright," Biochemical Pharmacology, 58:749-57, 1999.
4. B. Sinclair, "Glow power," The Scientist, 15:23, March 5, 2001.
Suppliers of Reporter Assay Systems
Packard BioScience Co.
PerkinElmer Life Sciences
Pierce Chemical Co.