Reporter gene
Encyclopedia
In molecular biology
, a reporter gene (often simply reporter) is a gene
that researchers attach to a regulatory sequence of another gene of interest in cell culture
, animals or plants. Certain genes are chosen as reporters because the characteristics they confer on organisms expressing them are easily identified and measured, or because they are selectable markers. Reporter genes are often used as an indication of whether a certain gene has been taken up by or expressed in the cell or organism population.
to be inserted into the cell or organism. For bacteria
or prokaryotic cells
in culture, this is usually in the form of a circular DNA molecule called a plasmid
. It is important to use a reporter gene that is not natively expressed in the cell or organism under study, since the expression of the reporter is being used as a marker for successful uptake of the gene of interest.
Commonly used reporter genes that induce visually identifiable characteristics usually involve fluorescent and luminescent proteins. Examples include the gene that encodes jellyfish green fluorescent protein
(GFP), which causes cells
that express it to glow green under blue light, the enzyme luciferase
, which catalyzes a reaction with luciferin
to produce light, and the red fluorescent protein from the gene dsRed. The GUS gene has been commonly used in plants but luciferase and GFP
are becoming more common.
A common reporter in bacteria is the LacZ
gene, which encodes the protein beta-galactosidase
. This enzyme causes bacteria expressing the gene to appear blue when grown on a medium that contains the substrate analog X-gal
. An example of a selectable-marker which is also a reporter in bacteria is the chloramphenicol acetyltransferase
(CAT) gene, which confers resistance to the antibiotic chloramphenicol
.
and transformation
- two ways of expressing a foreign or modified gene in an organism - are effective in only a small percentage of a population subjected to the techniques. Thus, a method for identifying those few successful gene uptake events is necessary. Reporter genes used in this way are normally expressed under their own promoter independent from that of the introduced gene of interest; the reporter gene can be expressed constitutively (that is, it is "always on") or inducibly with an external intervention such as the introduction of IPTG in the β-galactosidase system. As a result, the reporter gene's expression is independent of the gene of interest's expression, which is an advantage when the gene of interest is only expressed under certain specific conditions or in tissues that are difficult to access.
In the case of selectable-marker reporters such as CAT, the transfected population of bacteria can be grown on a substrate that contains chloramphenicol. Only those cells that have successfully taken up the construct containing the CAT gene will survive and multiply under these conditions.
into a single messenger RNA
molecule. The mRNA is then translated into protein. In these cases it is important that both proteins be able to properly fold
into their active conformations and interact with their substrates despite being fused. In building the DNA construct, a segment of DNA coding for a flexible polypeptide linker region is usually included so that the reporter and the gene product will only minimally interfere with one another.
, which aims to identify proteins that natively interact with one another in vivo
.
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
, a reporter gene (often simply reporter) is a gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
that researchers attach to a regulatory sequence of another gene of interest in cell culture
Cell culture
Cell culture is the complex process by which cells are grown under controlled conditions. In practice, the term "cell culture" has come to refer to the culturing of cells derived from singlecellular eukaryotes, especially animal cells. However, there are also cultures of plants, fungi and microbes,...
, animals or plants. Certain genes are chosen as reporters because the characteristics they confer on organisms expressing them are easily identified and measured, or because they are selectable markers. Reporter genes are often used as an indication of whether a certain gene has been taken up by or expressed in the cell or organism population.
Common reporter genes
To introduce a reporter gene into an organism, scientists place the reporter gene and the gene of interest in the same DNA constructDNA construct
A DNA construct is an artificially constructed segment of nucleic acid that is going to be "transplanted" into a target tissue or cell...
to be inserted into the cell or organism. For bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
or prokaryotic cells
Prokaryote
The prokaryotes are a group of organisms that lack a cell nucleus , or any other membrane-bound organelles. The organisms that have a cell nucleus are called eukaryotes. Most prokaryotes are unicellular, but a few such as myxobacteria have multicellular stages in their life cycles...
in culture, this is usually in the form of a circular DNA molecule called a plasmid
Plasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
. It is important to use a reporter gene that is not natively expressed in the cell or organism under study, since the expression of the reporter is being used as a marker for successful uptake of the gene of interest.
Commonly used reporter genes that induce visually identifiable characteristics usually involve fluorescent and luminescent proteins. Examples include the gene that encodes jellyfish green fluorescent protein
Green fluorescent protein
The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...
(GFP), which causes cells
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
that express it to glow green under blue light, the enzyme luciferase
Luciferase
Luciferase is a generic term for the class of oxidative enzymes used in bioluminescence and is distinct from a photoprotein. One famous example is the firefly luciferase from the firefly Photinus pyralis. "Firefly luciferase" as a laboratory reagent usually refers to P...
, which catalyzes a reaction with luciferin
Luciferin
Luciferins are a class of light-emitting biological pigments found in organisms that cause bioluminescence...
to produce light, and the red fluorescent protein from the gene dsRed. The GUS gene has been commonly used in plants but luciferase and GFP
Green fluorescent protein
The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...
are becoming more common.
A common reporter in bacteria is the LacZ
Lac operon
The lac operon is an operon required for the transport and metabolism of lactose in Escherichia coli and some other enteric bacteria. It consists of three adjacent structural genes, lacZ, lacY and lacA. The lac operon is regulated by several factors including the availability of glucose and of...
gene, which encodes the protein beta-galactosidase
Beta-galactosidase
β-galactosidase, also called beta-gal or β-gal, is a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides. Substrates of different β-galactosidases include ganglioside GM1, lactosylceramides, lactose, and various glycoproteins...
. This enzyme causes bacteria expressing the gene to appear blue when grown on a medium that contains the substrate analog X-gal
X-gal
X-gal is an organic compound consisting of galactose linked to a substituted indole. The compound was synthesized by Jerome Horwitz and collaborators in Detroit, MI, in 1964. The formal chemical name is often shortened to less accurate but also less cumbersome phrases such as bromochloroindoxyl...
. An example of a selectable-marker which is also a reporter in bacteria is the chloramphenicol acetyltransferase
Chloramphenicol acetyltransferase
Chloramphenicol acetyltransferase is a bacterial enzyme that detoxifies the antibiotic chloramphenicol and is responsible for chloramphenicol resistance in bacteria. This enzyme covalently attaches an acetyl group from acetyl-CoA to chloramphenicol, which prevents chloramphenicol from binding to...
(CAT) gene, which confers resistance to the antibiotic chloramphenicol
Chloramphenicol
Chloramphenicol is a bacteriostatic antimicrobial that became available in 1949. It is considered a prototypical broad-spectrum antibiotic, alongside the tetracyclines, and as it is both cheap and easy to manufacture it is frequently found as a drug of choice in the third world.Chloramphenicol is...
.
Transformation and transfection assays
Many methods of transfectionTransfection
Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
and transformation
Transformation (genetics)
In molecular biology transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material from its surroundings and taken up through the cell membrane. Transformation occurs naturally in some species of bacteria, but it can...
- two ways of expressing a foreign or modified gene in an organism - are effective in only a small percentage of a population subjected to the techniques. Thus, a method for identifying those few successful gene uptake events is necessary. Reporter genes used in this way are normally expressed under their own promoter independent from that of the introduced gene of interest; the reporter gene can be expressed constitutively (that is, it is "always on") or inducibly with an external intervention such as the introduction of IPTG in the β-galactosidase system. As a result, the reporter gene's expression is independent of the gene of interest's expression, which is an advantage when the gene of interest is only expressed under certain specific conditions or in tissues that are difficult to access.
In the case of selectable-marker reporters such as CAT, the transfected population of bacteria can be grown on a substrate that contains chloramphenicol. Only those cells that have successfully taken up the construct containing the CAT gene will survive and multiply under these conditions.
Gene expression assays
Reporter genes can also be used to assay for the expression of the gene of interest, which may produce a protein that has little obvious or immediate effect on the cell culture or organism. In these cases the reporter is directly attached to the gene of interest to create a gene fusion. The two genes are under the same promoter elements and are transcribedTranscription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
into a single messenger RNA
Messenger RNA
Messenger RNA is a molecule of RNA encoding a chemical "blueprint" for a protein product. mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes. Here, the nucleic acid polymer is translated into a polymer of amino acids: a protein...
molecule. The mRNA is then translated into protein. In these cases it is important that both proteins be able to properly fold
Protein folding
Protein folding is the process by which a protein structure assumes its functional shape or conformation. It is the physical process by which a polypeptide folds into its characteristic and functional three-dimensional structure from random coil....
into their active conformations and interact with their substrates despite being fused. In building the DNA construct, a segment of DNA coding for a flexible polypeptide linker region is usually included so that the reporter and the gene product will only minimally interfere with one another.
Promoter assays
Reporter genes can be used to assay for the activity of a particular promoter in a cell or organism. In this case there is no separate "gene of interest"; the reporter gene is simply placed under the control of the target promoter and the reporter gene product's activity is quantitatively measured. The results are normally reported relative to the activity under a "consensus" promoter known to induce strong gene expression.Further uses
A more complex usage of reporter genes on a large scale is in two-hybrid screeningTwo-hybrid screening
Two-hybrid screening is a molecular biology technique used to discover protein–protein interactions and protein–DNA interactions by testing for physical interactions between two proteins or a single protein and a DNA molecule, respectively.The premise behind the test is the activation of...
, which aims to identify proteins that natively interact with one another in vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
.
External links
- Research highlights and updated information on reporter genes.
- Staining Whole Mouse Embryos for β-Galactosidase (lacZ) Activity