REPORTER GENE ASSAY TECHNIQUE

Bioluminescence is simply defined as the emission of light from a biological source or living organisms. It is a type of luminescence that is enzymatically catalyzed, and in which the source of the light is from a biological entity such as the firefly (Figure 1). Certain microorganisms (including bacteria, fungi and algae), jellyfish and crustaceans also emit light. Luminescence is the emission of light energy as a result of chemical reaction; and it can occur in both living organisms and non-living organisms. Light emission from a biological source as aforementioned is generally known as bioluminescence while the emission of light from a non-biological source due to a chemical reaction is known as chemiluminescence. All bioluminescent organisms contain luciferin and luciferase which are both the major biochemical components of the bioluminescent activity. Luciferin is the light-emitting compound while luciferase is the enzyme (specifically an oxidoreductase) which catalyzes the oxidation of luciferin to form an energized-state which in turn produces light in the organism. The principle of light emission from both biological and non-biological sources has several biological applications including but not limited to their usage in detecting experimental changes in biological assays such as in reporter gene assay.

Reporter gene assay is a molecular biology technique that is used to evaluate the expression and regulatory potential of an unknown gene sequence. The reporter gene assay is generally used to study the expression of a gene; and it has applications in medical, pharmaceutical, biomedical and other molecular biology applications. This technique of reporter gene assay has been widely used for the investigation of gene regulation including gene expression and other important cellular activities in living systems. Reporter gene assay is also used to decipher the factors that control the expression of the gene of interest. Since a gene is known to contain two functional parts viz: the coding region and the promoter region, it is possible to evaluate the expression of an unknown gene by fusing or linking one part of the gene (especially the promoter region) to an easily detectable reporter gene (e.g. luciferase, the gene that codes for or controls the firefly bioluminescence activity). The coding region (which is the DNA sequence) is the part of the gene that specifies the protein to be made while the promoter region is the regulatory element of the gene that controls the transcription of the coding region. Beta (β)-galactosidase, β-glucuronidase, Green Fluorescent Protein (GFP) and luciferase are some examples of reporter genes.

By linking the promoter region of a particular gene of interest (either from prokaryotic or eukaryotic origins) to a reporter gene (e.g. luciferase) that is easily detectable, the expressed reporter gene proteins can be evaluated by a variety of assaying techniques such as absorbance and fluorescence. The enzymatic activity of the reporter genes especially luciferase is absent in most living cells, and thus its utilization in the assaying of gene expression is very sensitive and it provides a rapid means of measuring the expressed reporter gene protein. When a gene codes for the production of a protein molecule that has little effect on the host organism (or in cell culture plates) i.e. when the gene of interest is not conspicuously expressed, such a gene of interest can be assayed by coupling it to another gene (in this case: a reporter gene) in order to measure the level of its expression. After the fusion of the reporter gene with the promoter region of the gene of interest, a gene construct (bearing both genetic materials with the same promoter elements) is formed and this gene construct is transferred into a host cell (e.g. bacteria, plant or animal cell) through a genetic transfer method (Figure 2).

Figure 1: An illustration of the firefly (Photinus pyralis). In the firefly, chemical energy is converted to light energy through the help of adenosine triphosphate (ATP) which generates the energy that catalyzes the chemical reaction in the organism. The oxidation of luciferin by luciferase is also stimulated by oxygen and magnesium aside the input of ATP. Luciferin (the light-emitting compound), a complex carboxylic acid and the major biochemical component of bioluminescence in fireflies is broken-down or enzymatically cleaved by the enzyme, luciferase; and light is emitted by the firefly in the process.

Luciferase activity is usually measured using a piece of equipment known as luminometer (Figure 3). The gene construct or expression vector as it can also be called is usually formed by the process of cloning. Transfection and transformation are typical examples of genetic transfer techniques that can be employed to transfer the reporter construct or gene construct formed. The gene construct can also be injected directly into the host cell(s) via gene gun or any other molecular technique that can facilitate the introduction of genetic materials into a host cell. Inside the host cell, the gene construct undergoes transcription and translation to produce a protein molecule which is then measured to determine the activity of the regulatory protein i.e. the expressed protein molecule encoded by the gene of interest. Aside determining or measuring the activity of the regulatory protein itself, the enzymatic activity of the reporter protein can also be measured and this can be used to evaluate the gene expression of the cloned gene construct.

Figure 2: Illustration of luciferase reporter gene assay. With reporter gene assay, scientists can unravel and have answers to questions that surround the regulation of a gene and how genes are also expressed in a given cell (inclusive of microbial, plant, animal and human cells); and these techniques (i.e. the reporter gene assay) has tremendous applications in biomedical, medical and biopharmaceutical or pharmaceutical research.

LUMINOMETER

Luminometers are generally used to measure the experimental results of a luciferase assay. A luminometer (Figure 3) can be used for a wide variety of assays including luciferase assays, ATP assays, cellular luminescence, gene reporter assays, enzyme activity and cell-based assays for drug discovery. Luminometer is a programmable multiprocessor instrument with dual injectors capable of user-defined, variable volume reagent additions.
The features of the luminometer are as follows:
• The luminometer stores up to 40 separate assay programs
• It has a special high-sensitivity, low-noise detection system
• It is capable of functioning in various operating modes including carrying out statistical
analysis and kinetic measurement of samples
• The luminometer allows sensitive measurements of luciferase activity using the enhanced Luciferase Assay Kit, or other luminescent and chemiluminescent assays
Figure 3. Illustration of a luminometer.

References

Simon T.M, Allard and Kevin Kopish (2008). Luciferase Reporter Assays: Powerful, Adaptable Tools for Cell Biology Research. Cell Issues, 21:23-26. www.promega.com

Koo J, Kim Y, Kim J, Yeom M, Lee I.C and Nam H.G (2007). A GUS/Luciferase Fusion Reporter for Plant Gene Trapping and for Assay of Promoter Activity with Luciferin-Dependent Control of the Reporter Protein Stability. Plant and Cell Physiology 48(8): 1121–1131.

Ramawat K.G and Goyal S (2008). Comprehensive Biotechnology. Fourth revised edition. S.Chand and Company Ltd, Ram Nagar, New Delhi, India.

Brian Robert Shmaefsky (2006). Biotechnology 101. Greenwood Publishing Group, Inc, USA. Pp. 1-273.

Bourgaize  D,  Jewell  T.R  and  Buiser  R.G (1999). Biotechnology: Demystifying the Concepts. Pearson Education, San Francisco, CA.

Bains W (1998). Biotechnology: From A to Z. 2nd ed. Oxford University Press, New York, USA.

 

 

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