Reporter Gene Assays
Reporter genes have become an invaluable tool in studies of gene expression. They are widely used in biomedical and pharmaceutical research and also in molecular biology and biochemistry.
A gene consists of two functional parts: One is a DNA-sequence that gives the information about the protein that is produced (coding region). The other part is a specific DNA-sequence linked to the coding region; it regulates the transcription of the gene (promoter). The promoter is either activating or suppressing the expression of the gene.
The purpose of the reporter gene assay is to measure the regulatory potential of an unknown DNA-sequence. This can be done by linking a promoter sequence to an easily detectable reporter gene such as that encoding for the firefly luciferase.
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.
|Secreted alkaline phosphatase (SEAP)||+||+|
|Green Fluorescent Protein (GFP)||+|
Luciferase Reporter Gene Assay
The advantages of a luciferase assay are the high sensitivity, the absence of luciferase activity inside most of the cell types, the wide dynamic range, rapidity and low costs.
The most versatile and common reporter gene is the luciferase of the North American firefly Photinus pyralis. The protein requires no posttranslational modification for enzyme activity; it is not even toxic in high concentration (in vivo) and can be used in pro- and eukaryotic cells. The firefly luciferase catalyzes the bioluminescent oxidation of the luciferin in the presence of ATP, Magnesium and Oxygen.
Dual LuciferaseTM Reporter Gene Assay
The Dual-LuciferaseTM 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.