A gene consists of two main functional elements: the first consists of a coding DNA sequence that provides information about the protein produced. Second, a specific promoter sequence linked to the coding region that regulates the transcription of the gene. The promoter serves to activate or suppress the expression of the gene as required.
The main purpose of the reporter gene assay is to investigate the promoter of a gene of interest, i.e. the regulation of its expression. This can be done by linking the promoter of interest to an easily detectable gene, such as the gene for firefly luciferase, which catalyzes a reaction that produces light.
Typically, the cells are exposed to different factors or conditions the effect of which can be easily tracked by measuring changes in light emission.
Common reporter genes are β-galactosidase, β-glucuronidase and luciferase. Various detection methods (see below) are used to measure expressed reporter gene protein. These include luminescence, absorbance and fluorescence.
Assays based on luminescence are very popular for several reasons:
- they have a high sensitivity between 10 and 10,000 times higher than methods based on absorption or fluorescence, depending on the specific assay and reporter used.
- most cell types do not have endogenous luciferase activity
- luminescence assays have a large dynamic range
- they are quick to perform
- their costs are relatively low
Luminescent reporter assays are measured using a.
Reporter genes by detection method
|Secreted alkaline phosphatase (SEAP)|
|Green Fluorescent Protein (GFP)|
The most versatile and common reporter gene is the luciferase from the North American firefly Photinus pyralis. The protein requires no post-translational modification for enzyme activity. It is not toxic in-vivo even in high concentrations and can be used both in prokaryotic and eukaryotic cells. The firefly luciferase catalyzes the bioluminescent oxidation of the luciferin in the presence of ATP, Magnesium and Oxygen.
Dual-Luciferase® Reporter Gene Assay
The Dual-Luciferase® Reporter (DLR) Assay System contains two different luciferase reporter enzymes that are expressed simultaneously in each cell. The Firefly luciferase and the Renilla (sea pansy) luciferase can discriminate between their respective bioluminescence substrates and do not cross-activate. The Firefly luciferase is controlled by the promoter of interest and the Renilla luciferase by a promoter that gives a stable expression. This way the expression of the Renilla luciferase can be used as an internal control to compensate for any changes in cell number, transfection efficiency, and other errors. While this is very convenient, caution must be exercised to ensure that none of the conditions assayed modifies the expression of the Renilla luciferase, as this could lead to false conclusions.
In recent years other luciferases have been used for reporter gene assays, such as Gaussia, Cypridina or NanoLuc® luciferases. These so-called “novel” luciferases are up to 1000 times brighter than Firefly or Renilla luciferases and usually have other advantages. For example, improved stability, smaller size, and extracellular secretion.
Application Notes related to Reporter Genes
DLR Assay (Promega) with the Orion II Dual Luciferase Reporter Assay (Promega) with the Orion II Microplate Luminometer
PDF | 414.9 KB
Monitoring of Renilla Luciferase Activities in-vitro and in-vivo The expression of a novel EnduRen™ and ViviRen™ Renilla luciferase reporter gene (Promega) in vitro and in vivo is explored
PDF | 385.0 KB
Bioluminescence Imaging using NightOWL Bioluminescence Imaging using NightOWL LB 981 NC 100. Cells expressing luciferase gene under the control of a constitutive promoter were used as a model of in vivo proliferation of cancer cells.
PDF | 307.0 KB
Luciferin bioavailability in mice during in-vivo imaging To determine the optimal luciferase activity detection time, time course experiments were performed.
PDF | 315.7 KB
Circadian Clock with NightSHADE Getting to Know The Circadian Clock and Plant Growth With NightSHADE
PDF | 170.2 KB
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