Gene Analyses--Sunny Side Up!

Chemiluminescent Gene Reporter Systems Features of Luminometers How is the expression of a given gene controlled? That is a question asked of many in the biological and medical sciences. "Reporter genes," which encode quantifiable enzymes or proteins, are often employed by researchers seeking to better understand how the expression of genes of interest is controlled. In these assays, the coding region of the gene under study is replaced with the sequence that encodes the reporter gene. Reporter gene constructs contain one or more gene regulatory elements being analyzed; the structural sequence of the reporter gene, and sequences required for the formation of functional mRNA. Quantification of reporter gene products gives an indirect measure of the amount of gene expression obtained in a given set of conditions. Gene expression is controlled by the flanking, nontranslated "cis-acting" sequences, as well as by DNA-binding proteins and other factors that are said to act in "trans." Reporter gene assays can be used to fine-map cis elements such as promoters and enhancers and to help identify trans factors and gene-regulation mechanisms. A reporter gene must fulfill several basic criteria in order to be useful. A simple, cost-effective assay for quantifying the reporter protein or activity should be available, and the amount of reporter gene protein must correspond to the amount of reporter gene mRNA transcribed. Endogenous protein or enzyme activity must be absent or minimal in target cells. The assay should also have a broad linear range to enable analyses of both large and small changes in gene expression. Further, expression of the reporter should not alter the physiology of the recipient cells. An additional concern is the relative sensitivity of the assay. Sensitivity becomes an issue when attempting to analyze low levels of gene expression, especially in cell lines that transfect poorly. The sensitivity of each reporter gene assay does not only depend on the sensitivity of the detection method itself. It is also a function of numerous cell-line-specific factors, including reporter mRNA and protein turnover and endogenous (background) levels of the reporter activity. Commonly used detection techniques use radioisotopic, colorimetric, fluorometric, or chemiluminescent enzyme substrates. Because of their exquisite sensitivity, chemiluminescent assays for detecting reporter gene activities are favored by increasing numbers of researchers. These assays do not have the background or protein-quenching problems that sometimes plague fluorescent methods. Postdoctoral fellows, graduate students, and technicians will be thrilled to learn that chemiluminescent assays are reportedly less labor intensive than some of the alternatives. As a bonus, these nonisotopic assays don't have the disposal bugaboos and general aggravation associated with the more hazardous radioactive techniques. If you want to learn more about some of the chemiluminescent detection systems commonly used in reporter gene assays, read on! Packard's TopCount NXT microplate scintillation and luminescence counter Cloning of the firefly luc gene provided the first nonisotopic genetic reporter system with widespread utility in mammalian cells. A burst of green light, centering at 563 nm, is emitted as firefly luciferase catalyzes the ATP-dependent oxidation of luciferin. The first-developed firefly luciferase assays were performed in the absence of coenzyme A. In the absence of the coenzyme, the luciferase-generated flashes decay in less than one second. Light signals with such super-quick "flash kinetics" can be measured using a luminometer equipped with an autoinjection device. The injector facilitates rapid mixing of reaction components. Alternatively, a liquid scintillation counter can be employed to record the light signals, albeit with lower sensitivity. These original luciferase assays garnered quite a bit of interest in the research community because they were 10-1,000 times more sensitive than conventional radioactive chloramphenicol acetyltransferase (CAT) assays. However, the earlier assays did have their difficulties, most of which were due to the flash kinetics of the photon emission. In response, several different protocols that led to an easily measured, sustained glow of light were developed. If you are a "high throughput" screener evaluating many samples simultaneously, then glow kinetics aren't just more convenient--they are necessary. For these applications, "flash-type kits will not work because the signal will be gone before the samples are read," explains Keith Osiewicz, associate product manager, Packard Instrument Company. "But with glow type signals they can sit in stackers for hours before being read with little signal degradation," adds Osiewicz. Today, most luciferase fans use an improved glow-type assay that includes coenzyme A in the reaction mixture. The resultant light output is longer and more intense, producing an assay that is not only easier to perform but more sensitive as well. Native firefly luciferase contains a peroxisome targeting sequence. As a result, luciferase expressed from conventional vectors has a half-life of only about 3 hours when expressed in most mammalian cells. The rapid turnover of firefly luciferase expressed from these first-generation vectors isn't always an overwhelmingly bad feature. For example, this relatively short-lived form of luciferase is a good candidate reporter for inducible systems. Need your luciferase to hang around longer? You are in luck. Newer constructs lacking the peroxisome targeting sequence are now available. Firefly luciferase expressed from these more modern vectors localizes to the cytoplasm, and consequently stays functional much longer. Firefly luciferase has a broad linear range, spanning seven to eight orders of magnitude. Promega offers an array of vectors for luciferase gene reporter assays as well as kits for detecting luciferase activity. Detection kits for luciferase reporter gene assays are also available from several other companies (Chemiluminescent Gene Reporter Systems Table). Luc-Screen™ assays were performed with 28,000 NIH/3T3 cells transiently transfected with pGL-3 luciferase vector in the presence of culture medium with (closed circles) or without (open circles) phenol red. The light signal from Luc-Screen assays is stable for at least 90 minutes in either the absence or presence of phenol red, which causes a two - three fold reduction in signal intensity. Image provided by Tropix. Luciferases from species other than firefly have also been cloned and used in reporter gene assays. These enzymes are structurally unrelated to firefly luciferase and catalyze different bioluminescent reactions. Perhaps the most widely used of these is the sea pansy (Renilla reniformis) luciferase. Under standard assay conditions, this luciferase catalyzes the oxidation of coelenterazine to produce a flash of blue light at 482 nm. The assay conditions can be changed to alter the kinetics of the reaction, resulting in an extended, readily measured light signal. Promega's Dual-LuciferaseTM Reporter Assay System uses the sequential measurement of the firefly, then the Renilla activity. (Tropix Dual Light® uses sequential measurement of firefly luciferase, then ß-galactosidase.) The two activities are distinguished temporally, not by wavelength. In this assay, firefly luciferase or Renilla activity is used as the reporter gene or the control interchangeably. There is no prerequisite for either of these.1 The cDNA encoding secreted alkaline phosphatase (SEAP) is useful for investigating the function of elements in gene-expression regulation. In nature, alkaline phosphatase is a membrane-bound protein. SEAP is a genetically engineered, truncated form of placental alkaline phosphatase. This variant lacks a membrane-anchoring domain. Unlike wild-type placental alkaline phosphatase, SEAP is secreted from the cell. In contrast to other alkaline phosphatase isoforms, placental alkaline phosphatase and SEAP are extremely heat stable and are resistant to the phosphatase inhibitor L-homoarginine. Endogenous alkaline phosphatase activity in transfected cells can be minimized by pretreatment of samples at 65ºC and incubation with the inhibitor. The fact that SEAP is secreted from the cell makes this method attractive to many researchers. SEAP activity can be readily detected in a sample of the culture medium, leaving the cell population intact for further investigation. The first-developed SEAP chemiluminescent assays were two-step, indirect measures of the phosphatase. In these assays, SEAP first hydrolyzed D-luciferin-O-phosphate to form D-luciferin. The newly formed D-luciferin then served as the substrate for the second, luciferase-catalyzed, chemiluminescent step. The most sensitive chemiluminescent SEAP assays are those in which the phosphatase itself catalyzes the luminescent hydrolysis of 1,2-dioxetane substrates such as CSPD®. (For a review of chemiluminescent alkaline phosphatase substrates, see reference 2.) Dephosphorylation of 1,2-dioxetane substrates results in the emission of blue light (470 nm) with a sustained, glow type of luminescence. Chemiluminescent SEAP assays enjoy a broad linear range (six to seven orders of magnitude dynamic range, from femtogram to nanogram levels of enzyme). However, keep in mind that SEAP assays are probably not suitable for cells from tissues that express even very low levels of placental alkaline phosphatase. HeLa cells for example, as well as some other cancer cell lines may produce unacceptably high levels of phosphatase activity.3 Complete kits containing vectors and detection reagents needed for SEAP reporter gene assays are manufactured for CLONTECH and ICN by Tropix. Those nifty yellow colorimetric assays that use ONPG as a substrate have long been ß-galactosidase's (ß-gal) claim to fame. Some new tricks have been used to put this old workhorse in the spotlight again. The development of 1,2-dioxetane substrates for ß-gal have made this enzyme a viable candidate reporter. Chemiluminescent ß-gal assays have an extraordinarily broad linear range (five to six orders of magnitude). Chemiluminescent ß-gal-based reactions are frequently used to normalize the variability of other reporter assays. The Dual-Light® Reporter Gene Assay System from CLONTECH and Roche Molecular Biochemicals' chemiluminescent detection reagents are both 1,2-dioxetane compounds manufactured by Tropix. Vectors and detection kits for the more general chemiluminescent ß-gal reporter gene assay are also available from ICN. The bacterial ß-glucuronidase (GUS) gene is an old-time favorite of plant biologists. GUS can be used as a reporter to study gene expression in both plants and mammalian cells. Because most higher plants lack endogenous GUS activity, this enzyme has proven particularly advantageous for use in those species. As is the case with ß-gal, both colorimetric and fluorescent assays have been widely used. And guess what? Chemiluminescent 1,2-dioxetane-based assays have also been developed for GUS. (Surprised?) These GUS-based assays have phenomenal linear ranges that span six orders of magnitude. GUS detection systems for plant as well as mammalian systems are available from both ICN and Tropix. Turner Designs' TD-20/20 Luminometer. A number of companies sell luminometers useful for measuring chemiluminescent signals in reporter gene assays. A table of features of luminometers is available on our Web site. The Turner Designs TD-20/20 luminometer is a versatile instrument that can measure light output from a wide variety of containers, including test tubes, microfuge tubes, culture dishes, and scintillation vials. Programmable autoinjectors needed for flash chemiluminescent reactions are available. This company, based in Sunnyvale, Calif., has spent some time making sure that its products are readily usable with other commercially available products. For example, the TD-20/20 Luminometer comes standard with software for conducting Promega's Dual-Luciferase assays. You can even order the luminometer directly from the Promega catalog! Turner Designs' literature about this instrument also includes a protocol for using the instrument with CLONTECH's Great EscAPe SEAP Reporter System. PharMingen now offers the MonolightTM 3010, a programmable luminometer with dual injectors. This instrument comes preprogrammed for several different chemiluminescent applications, including ß-galactosidase-, luciferase-, and Dual-Luciferase Reporter- assays. The unit features a dual measuring chamber rotating turret. This enables one to measure one sample while the next sample is being loaded. The Monolight 3010 also offers a software interface that allows storage of protocols and instrument settings in a "credit card" format. Future software upgrades will also be made available through this mechanism. The staff at Laboratory Technologies is excited about ACCULYTE 10TM, which features a novel light-collection chamber. They claim that this chamber delivers up to 10 times the sensitivity of other luminescent analyzers and that it is ideally suited to maximize sensitivity in both glow and flash types of luminescence. The ACCULYTE 10 user can select no, one, two, or three injectors. The true lab sophisticate will love the fact that these injectors can be fired individually, sequentially, or at timed intervals. This provides the ability to perform multireagent flash reactions. The unit comes with an array of software for data analysis and transfer. MGM Instruments supplies luminometers to a number of manufacturers of clinical assays, as well as members of the academic research community for various applications, especially reporter gene assays. The most popular model is the OPTOCOMP I, an economically priced manual luminometer. The OPTOCOMP II is capable of running up to 250 samples automatically and includes software for performing Promega's DualLuciferaseTM assays. Optional reagent injectors are available with both the OPTOCOMP I and the OPTOCOMP II models. These instruments also feature menu-driven software with a number of data analysis options and built-in thermal printers. For the budget-minded, the company offers OPTOCOMP P, a portable, battery-powered luminometer. (Yes, you read that correctly). MGM Instruments says that the OPTOCOMP P is as sensitive as its other luminometers, and is an economical instrument for researchers who do not need injectors. Representatives at MGM are especially proud of the company's long-standing "try before you buy" policy. The company will provide any of their instruments to researchers for a no-obligation evaluation period of one month. If small appeals to you, check out the LumiOneTM luminometer from Bioscan, Washington, D.C. This Lilliputian device takes up less than 1 square foot of bench space. The company also offers the LumiScintTM, a benchtop liquid scintillation counter/luminometer combo. Another compact benchtop photometer is the Lumi-Tec, from St. John Associates. Test tube and flow cell models are available. The test tube instrument uses either 6 x 50 mm disposable glass cuvettes or 12 x 50 mm polystyrene cuvettes. The cuvette is placed into the light measurement chamber by way of a sliding drawer with a built-in shutter. This provides easy access to the disposable cuvette while preventing stray light from entering the photodetection system. Reagent addition and mixing are accomplished by syringe injection through a light-tight septum injection port. The standard flow instruments have 70 µl, 1 meter coil flow cells placed directly in front of the photomultiplier detector. Other cell volumes and lengths are available on request. Flow cells can be furnished with or without an integral mixing tee for reagent addition. Packard Instrument Company says its LumiCount Microplate Luminometer offers superior sensitivity and affordability for reading sustained glow luminescent assays. This luminometer is designed for use with various microplate formats (6, 12, 24, 48, or 96 wells). Microplate assays are very useful for those who need to perform large numbers of reporter gene assays with minimal use of reagents. LumiCount features a wide detection wavelength range, from 300 nm to 650 nm. The instrument can be integrated with automatic liquid handlers or robotic automation systems. This instrument has a plate heater that can warm samples up to 45°C. Want to analyze plates for radioactivity and luminescent signals? Packard also offers TopCount NXT, which is a fully contained benchtop microplate scintillation counter and luminometer. This instrument has temperature control, albeit with the fairly narrow range of 19°-35°C. Both units use Windows-based software. Tropix is well known for having developed reagents for chemiluminescent assays. The company also offers the TR717TM Microplate Luminometer, which reads 96- or 384-well plates. TheTR717TM Microplate Luminometer is compatible with most robotics systems for sample preparation. The unit comes with user-friendly Windows-based software for data interpretation. Autoinjectors are optional. Models with injectors also include plate heaters that can warm samples up to 42°C. The MLXTM Microtiter® Luminescence Detection Systems from Dynex Technologies are geared toward measuring luminescence in 96-well plates. RevelationTM, the company's operating and data analysis software, is available in both Macintosh and Windows formats. The system can be configured with up to three dispensers, enabling one to perform multireagent flash reactions. This luminometer also features a broad temperature control range, from 5°-45°C. Four luminometers are currently offered by Rosys Anthos. Gottfried Griesmayr, product manager, says that all four members of the Lucy luminometer series are ideally suited for reporter gene detection and have a number of special features for this application. For example, the combined luminometric and photometric measurement mode enables the measurement of colorimetric labels. Dual- or multi-reporter assays can be performed by combining luminescent and colorimetric labels for different reporter enzymes in one assay. The software allows any combination of dispensing and measurement steps and includes a special mode for dual reporter assays such as the one sold by Promega. Lucy1 is a combined luminometer/photometer. It allows detection in the spectral range from 340 nm to 750 nm. A single or twin dispenser is optional. The control software is menu driven. Lucy1 also offers temperature control within the range of 15°-40°C. Lucy2 is an economical model that is controlled via an external personal computer (PC). Lucy3 is the stand-alone version of Lucy2, with identical measurement technology and built-in operation and data analysis. AutoLucy is a highly flexible, robotic plate processor and analyzer. It combines a robotic microplate processor and the Lucy luminometer/photometer. Freely programmable, it can access reagents from any container and uses multiple pipetting tips to efficiently assemble assays. Samples and microplates may be identified by barcode, and a robotic hand transports plates between optional modules such as incubators, stackers, washers, and shakers. A single measurement of 96 wells takes less than 40 seconds. Complex kinetic sequences can be set up by well, row, or whole plate. AutoLucy is controlled by an external PC and uses Windows software. EG & G Life Sciences' Flash 'n Glow EG&G Life Sciences supplies high performance tube luminometers from the portable Berthold junior to the high throughput Berthold AutoLumat. A new member of the range is the Berthold flash 'n glow. With a 30-place sample changer and up to three volume-adjustable injectors, it is ideal for reporter gene assay. The Berthold MicroLumatPlus is a dedicated and highly sensitive DLReadyTM plate luminometer accepting 96-well or 384-well plates. EG&G Life Sciences is known for multilabel plate counters with luminescence as one technology alongside others. The EG&G Life Sciences VICTOR2 is a fluorometer, time-resolved fluorometer, luminometer, and photometer in one instrument. It accepts plates with from one to 864 wells and supports both flash type and glow-type luminescence assays. Finally, the EG&G Life Sciences' MicroBeta is a liquid scintillation plate counter that can be used with most laboratory radioisotopes as well as with flash-type and glow-type luminescence reagents. The EG&G Life Sciences MicroBeta JET is, like the Berthold MicroLumat, DLReady. The focus of this article has been in vitro reporter gene assays. You should be aware that in vivo reporter gene assays are also available. These methods generally provide less quantitative information than do the in vitro assays, but they can yield information regarding the cell- or tissue-type specificity of gene regulatory elements and factors. Currently, Green Fluorescent Protein (GFP) assays are favored for in vivo applications. These assays are sensitive and allow for real-time analysis. Firefly luciferase has been used as a reporter in chemiluminescent in vivo gene reporter assays. Though luciferase does not offer real-time analysis, it merits consideration for use in other applications. Firefly luciferase activity can be detected in live cells by use of soluble luciferin derivatives that cross the plasma membrane. Once inside the cell, endogenous enzymes convert these substrates into luciferin, and the rest is history. A photolyzable "caged" version of luciferase is available from Molecular Probes. Caged luciferin appears to enter cells more efficiently than its forerunners and can be hydrolyzed to form free luciferin either by endogenous enzymes or by visible light. Chemiluminescent systems for reporter gene assays have many advantages. Keep in mind, however, a number of problems are associated with using reporter genes, regardless of the detection method chosen. For example, sequences within the coding region of a gene are sometimes involved in the regulation of its promoter. When you substitute the reporter gene for the structural gene of interest, you remove the promoter from those regulatory mechanisms. Different types of problems can arise if the reporter gene itself contains sequences that alter the function of the promoter under study. It is important to verify that regulatory events uncovered by use of a fusion gene actually occur with the native genes in vivo. The much-loved laboratory reference Current Protocols in Molecular Biology is a good starting place for those interested in learning more about reporter gene assays, their caveats, and alternatives. Some other good references are listed in the section below. D. Wilkinson, "Bring out the sunglasses: Promega's Dual-Luciferase Reporter Assay System," The Scientist, 12[16]:15, Aug. 17, 1998. D. Wilkinson, "A shot in the dark: products for the chemiluminescent detection of DNA," The Scientist, 12[10]:20, May 11, 1998. F.J. Benham et al., "Alkaline phosphatase expressionin human cell lines derived frm various malignancies," International Journal of Cancer, 27:637-44, 1981. S. Kain, S. Ganguly, "Overview of genetic reporter systems," Current Protocols in Molecular Biology, F.M. Ausubel, ed., Unit 9.6:1-12, 1995. I. Bronstein et al., "Chemiluminescent and bioluminescent reporter gene assays," Analytical Biochemistry, 219:169-81, 1994. J. Alam, J.L. Cook, "Reporter genes: application to the study of mammalian gene transcription," Analytical Biochemistry, 188:245-54, 1990. For more information about products mentioned in this article please circle Reader Service #73. The author can be contacted at deborahwilkinson@sprynet.com. Chemiluminescent Gene Reporter Systems Features of Luminometers

Gene Analyses--Sunny Side Up!

Mar 28, 1999

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