Reporter Gene Assays

Genetic reporter systems represent an extensive toolbox for the study of regulatory promoter and enhancer sequences as well as for the study of transcription factors. Regulatory sequences of interest are combined with a reporter construct of choice and are subsequently assayed in conjunction with relevant transcription factors.
If the reporter system is well chosen then the level of reporter gene expression will correlate with the transcriptional activity of the introduced transgenic factors. In order to assure such a correlation, it is important that the reporter gene does not disturb the metabolism of the transformed cells and that the gene is not endogenously expressed by the target cells creating background signals.
Expression of reporter genes can be measured by:

  • Enzyme activity assay of the expressed enzyme encoded by the reporter gene using chromo-, fluoro- or luminogenic substrates.
  • Immunological assay of the expressed protein encoded by the reporter gene (reporter gene ELISA).
  • Histochemical staining of cells or tissues typically to localize enzymatic activity ectopically expressed from reporter gene constructs in transformed cells.


A considerable number of reporter gene systems has been developed over the years. The most commonly used reporter gene systems include:

 

Chloramphenicol Acetyltransferase (CAT) Reporter System

Microbial chloramphenicol acetyltransferase (CAT) catalyzes the transfer of acetyl groups from acetyl coenzyme A to chloramphenicol and represents the oldest reporter gene system still in broad use today. The assay is performed by incubating transformed cells with chloramphenicol carrying a radioactive label. CAT activity can be assayed by autoradiography of lysate subjected to thin layer chromatography (TLC). Acetylated and non-acetylated chloramphenicol can readily be separated by TLC of lysate. Hence, the presence of labeled acetylated chloramphenicol correlates to the expression of CAT. In an effort to limit the use of radioactivity in the lab the method is rapidly being replaced by immunological detection of the CAT enzyme (ELISA).

Selection of Biosynth's HRP Substrates
Cat. No. Product Name
D-2000 3,5-Dichloro-2-hydroxybenzenesulfonic acid, sodium salt
D-2050 3,5-Dichloro-2-hydroxybenzenesulfonic acid, disodium salt
T-2100 3,3',5,5'-Tetramethylbenzidine, free base
T-2150 3,3',5,5'-Tetramethylbenzidine dihydrochloride
Selection of Biosynth's Alkaline Phosphatase Substrates
Cat. No. Product Name
C-5100 6-Chloro-3-indoxyl phosphate, p-toluidine salt
I-6250 3-Indoxyl phosphate, disodium salt
I-6300 3-Indoxyl phosphate, p-toluidine salt
M-5800 4-Methylumbelliferyl phosphate, free acid

Selection of Biosynth's Tetrazolium Salts
Cat. No. Product Name
N-8100 Nitrotetrazolium blue chloride
T-2300 Tetranitro blue tetrazolium chloride
T-2590 Tetrazolium blue chloride
T-2600 Tetrazolium violet
X-5000 XTT, sodium salt

LacZ Reporter Gene System


Colonies expressing lacZ appear blue on culture media containing X-gal (B-7150)

The prokaryotic beta-galactosidase encoded by lacZ from the lac operon catalyzes the hydrolysis of beta-galactosides, most prominently the hydrolysis of lactose. Galactosidase activity in lysates of transfected cells can be assayed via absorption of o-nitrophenolate from o-nitrophenyl-ß-D- galactoside (ONPG,N-4190), fluorescence of 4-methylumbelliferone (from 4-methyl-umbelliferyl-ß- galactopyranoside,M-5550), or via chemiluminescence of 1,2-dioxetan-galactopyranoside derivatives. The lacZ is often co-transfected together with other reporter constructs such as luciferase serving as an internal control.

The lacZ reporter gene is widely used as a convenient tool to identify recombined plasmids during molecular cloning procedures.
Since the complete lacZ gene is too large to be carried by a plasmid, cloning vectors contain only the alpha region of the lacZ gene. Expression of the alpha region of lacZ yields an inactive protein. However, the truncated protein encoded by the alpha region can combine into active beta-gal when complemented with a protein from lacZ lacking the alpha region.
Such “intragenic complementation” can be achieved if a plasmid carrying the alpha region of lacZ is inserted into a bacterial cell containing a mutated alpha region of the lacZ gene. Such cells will turn beta-gal positive if the plasmid is present.
DNA cloning into lacZ vectors occurs such that the fragment is likely to insert into the alpha lacZ sequence on the plasmid which renders the alpha-region dysfunctional. In this case intragenic complementation no longer occurs. Hence, cells carrying plasmids containing foreign DNA will remain beta-gal negative.
Beta-gal negative cells can readily be isolated from positive ones by incubation of bacteria on chromogenic culture media containing X-gal (5-bromo-4-chloro-3-indoxyl-beta-D-galactopyranoside, B- 7150) and IPTG (isopropyl-beta-D-thiogalactopyranoside, I-8000) followed by manual selection of the colorless colonies.

Selection of Biosynth's beta-Galactosidase Substrates
Cat. No. Product Name
B-7150 5-Bromo-4-chloro-3-indoxyl-beta-D-galactopyranoside
B-7200 5-Bromo-6-chloro-3-indoxyl-beta-D-galactopyranoside
B-8900 5-Bromo-3-indoxyl-beta-D-galactopyranoside
C-4990 4-Chloro-3-indoxyl-beta-D-galactopyranoside
H-7500 8-Hydroxyquinoline-beta-D-galactopyranoside
I-5660 3-Indoxyl-N-acetyl-beta-D-galactopyranoside
I-7600 5-Iodo-3-indoxyl-beta-D-galactopyranoside
I-8050 Isopropyl-beta-D-thiogalactopyranoside dioxane-grade
M-5550 4-Methylumbelliferyl-beta-D-galactopyranoside
N-2030 Naphthol AS-BI-beta-D-galactopyranoside
N-2255 1-Naphthyl-beta-D-galactopyranoside

Selection of Biosynth's beta-Galactosidase Inducers
Cat. No. Product Name
I-8000 Isopropyl-beta-D-thiogalactopyranoside dioxane free
M-3590 1-O-Methyl-alpha-D-galactopyranoside
M-3591 1-O-Methyl-beta-D-galactopyranoside
M-3600 1-O-Methyl-beta-D-glucuronic acid, sodium salt
M-4440 Methyl-beta-D-thiogalactopyranoside
M-6600 1-O-Methyl-beta-D-xylopyranoside
P-4250 Phenyl-beta-D-thiogalactopyranoside

GUS Reporter Gene System

GUS encodes the beta-glucuronidase enzyme from E. coli and it has been extensively used as a marker gene in transgenic plants. The main advantage of the GUS reporter gene system is the stable expression of GUS enzyme in plant cells. GUS activity can readily be detected by chromogenic or fluorogenic enzyme substrates. Typically, very little background signal is encountered because the intrinsic expression of endogenous beta-glucuronidase genes is not significant in plants. In addition, plant metabolism remains largely unaffected by the presence of E. coli beta-glucuronidase. Various beta-glucuronic acid substrates are available for detection of GUS expression. The most widely used fluorogenic substrate for detection of beta-glucuronidase activity in vitro is 4- methylumbelliferyl beta-D-glucuronide (MUG, 4-methylumbelliferyl-beta-D-glucuronic acid dihydrate, M-5700). For other applications the use of X beta-glucuronide (5-bromo-4-chloro-3-indoxyl-beta-D- glucuronic acid, cyclohexylammonium salt, B-7300), which yields an insoluble blue precipitate in the presence of beta-glucuronidase, has become standard.

Selection of Biosynth's Glucuronidase Substrates
Cat. No. Product Name
B-7300 5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid, cyclohexylammonium salt
B-7350 5-Bromo-6-chloro-3-indoxyl-beta-D-glucuronic acid, cyclohexylammonium salt
B-8902 5-Bromo-3-indoxyl-beta-D-glucuronic acid, cyclohexylammonium salt



Figure:  Transgenic tobacco seedlings, expressing GUS (beta-glucuronidase) under the control of the CaMV-35S promoter. The seedlings were stained using standard conditions with vacuum infiltration of either X- glcUA (B-7300), MagentaTM-beta-D-glcUA (B-7350) or SalmonTM-beta-D-glcUA (C-5050) at 1 mg/ml, with overnight incubation at 37°C. (Courtesy of Dr. R.A. Jefferson)

SEAP Reporter Gene System

Another secreted reporter protein is the SEAP (secreted alkaline phosphatase). This protein is quantified directly by measuring the enzyme activity in the supernatant of the culture medium. Fluorescence and chemiluminescence assays are available for detection.

Selection of Biosynth's Substrates for Alkaline Phosphatase
Cat. No. Product Name
B-7450 5-Bromo-4-chloro-3-indoxyl phosphate, disodium salt sesquihydrate
B-7452 5-Bromo-6-chloro-3-indoxyl phosphate, disodium salt monohydrate
B-7453 5-Bromo-6-chloro-3-indoxyl phosphate, disodium salt trihydrate
B-7500 5-Bromo-4-chloro-3-indoxyl phosphate, p-toluidine salt
B-7550 5-Bromo-6-chloro-3-indoxyl phosphate, p-toluidine salt
C-5100 6-Chloro-3-indoxyl phosphate, p-toluidine salt
I-6200 3-Indoxyl phosphate, bis(2-amino-2-methyl-1,3-propanediol) salt
I-6250 3-Indoxyl phosphate, disodium salt
I-6300 3-Indoxyl phosphate, p-toluidine salt
M-5800 4-Methylumbelliferyl phosphate, free acid
M-5850 4-Methylumbelliferyl phosphate, bis(2-amino-2-methyl-1,3-propanediol) salt
M-5900 4-Methylumbelliferyl phosphate, bis(cyclohexylammonium) salt
M-5920 4-Methylumbelliferyl phosphate, dilithium salt
M-5950 4-Methylumbelliferyl phosphate, disodium salt trihydrate

Luc Reporter Gene System

 
Photinus pyralis

In nature, the luciferase reaction occurs in the peroxisomes of a specialized light organ in fireflies (Photinus pyralis). The luciferase reaction emits a yellow-green light (560nm) and requires the co-factors ATP, Mg 2+ , O2 and the substrate luciferin. The glow is widely used as an assay for luciferase activity to monitor regulatory elements that control its expression. Luc is particularly useful as a reporter gene since it can be introduced into living cells and into whole organisms such as plants, insects, and even mammals. Luc expression does not adversely affect the metabolism of transgenic cells or organisms. In addition, the luc substrate luciferin is not toxic to mammalian cells, but it is water-soluble and readily transported into cells. Since luc is not naturally present in target cells the assay is virtually background-free. Hence, the luc reporter gene is ideal for detecting low-level gene expression. A second reporter system based on luciferase expressed by the ruc gene from Renilla (Renilla reniformis) has also become available. The activities of firefly and Renilla luciferase can be combined into a dual reporter gene assay.

Luciferase expression is measured by adding ATP and luciferin to cell lysates and then analyzing bioluminescence by spectroscopy or with a scintillation counter allowing for the detection of even a few hundred thousand enzyme molecules.

The development of camera based imaging systems to visualize luc gene expression in vivo has made rapid progress; most impressively, in the generation of transgenic mice carrying the luc gene. In some of these mice strains luc expression is triggered by intoxication or the inflammation processes. The effects of disease to mice as well as the results of therapy can be studied non-invasively and in real time (www.xenogen.com).
Luciferin was initially isolated by extraction from Photinus pyralis which is a tedious process. Despite manufacturing bulk quantities of luciferin chemically, commercial grade luciferin is still specified “synthetic” in contrast to the long-gone process of extraction from Photinus pyralis.

Selection Biosynth's Luciferase Substrates
Cat. No. Product Name
L-8080 Luciferase, American Firefly, natural, high purity
L-8200 D-Luciferin Firefly (synthetic)
L-8220 D-Luciferin Firefly, potassium salt (synthetic)
L-8280 L-Luciferin, potassium salt
L-8300 D-Luciferin 1-(4,5-dimethoxy-2-nitrophenyl)ethyl ester
L-8305 DL-Luciferin 1-(4,5-dimethoxy-2-nitrophenyl)ethyl ester

Ruc Reporter Gene System



Renilla luciferase serves as a marker protein in bioluminescent fusion constructs of the ruc gene. It is similar to the above described luc vectors which is useful in low-light imaging of gene expression and of regulation processes in living cells as well as in transgenic plants. Coelenterazine e is the best substrate for Renilla luciferase generating 40 % more light than native coelenterazine with a considerably higher initial light intensity.



The firefly luciferase gene (luc) has proven to be very useful in reporter gene research and drug discovery assays, when used with luciferin and ATP: firefly luciferase/luciferin emits light at 560 nm, Renilla luciferase/coelenterazine emits light at 475 nm. Because these two reporter systems emit light at quite different wave lengths, it is possible to use firefly luciferase/luciferin and Renilla luciferase/coelenterazine as a dual reporter system.

In dual reporter gene systems two distinct reporter enzymes can be measured within one sample, whereby the second reporter is often used as an internal control. Dual reporter luminescence assays preferably using firefly luciferase as the first reporter enzyme, have become very popular, e.g. for measuring transcriptional activity in studies of the structural or physiological basis of regulated gene expression.

Green Fluorescent Protein (GFP) Reporter Gene System

The green fluorescent protein from the Aequorea victoria jellyfish emits green fluoresence without the need for any enzyme or co-factors. Gene expression and protein localization is possible in situ and in vivo: no secondary transformation is required. Reporter gene expression can be qualitatively monitored live e.g. for the study of dynamic processes inside the cell. There are a number of GFP proteins in use. Selecting GFP with minimal overlap in light emission allows dual reporter gene studies, for instance, to localize different sites of expression or, the relative effects of different regulatory proteins.


Bioluminescence Resonance Energy Transfer (BRET)

Most recently, bioluminescence resonance energy transfer (BRET) detection systems have been introduced. These systems are based on excitation energy transfer from a bioluminescent donor molecule, e.g. Renilla luciferase to a fluorescent acceptor molecule, like green fluorescent protein (GFP). It is known that luciferase interacts rapidly with GFP in a highly specific manner to form an equilibrium complex responsible for this very efficient energy-transfer phenomenon.

Renilla luciferase as well as GFP are both attached to the proteins to be examined, e.g. by chemical synthesis or by use of genetic engineering methods. In the latter case the gene fusion constructs are used to transfect or transform cells and express the fusion proteins. In the presence of coelenterazine, a BRET signal is generated when the fusion proteins are associated.
Excellent discrimination of the ruc and GFP signals results when coelenterazine 400a is used as the ruc substrate.

As the efficiency of the non-radioactive energy transfer from the Renilla luciferase fusion construct to the GFP conjugate depends on steric effects, for instance, on the distance of the protein molecules and on their relative orientation, the method can be used to study protein-protein interaction as well as to screen for inhibition of such an interaction: in drug discovery applications or receptor-ligand studies. The new spectroscopic techniques and assay methods built on BRET are fascinating tools for investigation in vivo.

Human Growth Hormone (hGH) Reporter Gene System

The human growth hormone (hGH) encoded reporter protein is secreted into the culture medium by transfected cells. Thus no cell lysis is required for detection. Detection of the secreted hGH requires the use of hGH antibodies. Such antibodies used to be radiolabeled in order to allow detection of the antibody hGH complex. Today hGH is assayed by indirect ELISA. First, the hGH from the supernatant of the culture medium binds to the antibody on the plate. Subsequently, the bound hGH is detected in two steps via a digoxigenin-coupled anti-hGH antibody and a peroxidase-coupled anti- digoxigenin antibody. Bound peroxidase is quantified by incubation with a peroxidase substrate such as TMB (3,3',5,5'-tetramethylbenzidine,T-2100).



List of HRP Substrates
Cat. No. Product Name
D-2000 3,5-Dichloro-2-hydroxybenzenesulfonic acid, sodium salt
D-2050 3,5-Dichloro-2-hydroxybenzenesulfonic acid, disodium salt
T-2100 3,3',5,5'-Tetramethylbenzidine, free base
T-2150 3,3',5,5'-Tetramethylbenzidine dihydrochloride
List of Tetrazolium Reagents
Cat. No. Product Name
I-7650 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride
N-8100 Nitrotetrazolium blue chloride
T-2300 Tetranitro blue tetrazolium chloride
T-2590 Tetrazolium blue chloride
T-2600 Tetrazolium violet
T-6940 2,3,5-Triphenyltetrazolium chloride reagent-grade
X-5000 XTT, sodium salt


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