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. Genetic reporters can be used to study a wide range of biological areas, from gene expression and control to cell signaling and drug discovery.
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 ß-galactosidase, ß-glucuronidase and luciferase. Various detection methods (see below) are used to measure expressed reporter gene protein. These include luminescence, absorbance and fluorescence.
Reporter Detection Method
Luminescence Fluorescence Absorb
Luciferase + + -
ß-Galactosidase (GUS) + + +
ß-Glucuronidase (b-Gal) + + -
(SEAP) + - +
Green Fluorescent Protein (GFP) - + -
Secreted placental alkaline phosphatase (SEAP)
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.
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
LacZ Reporter Gene System
GUS Reporter Gene System
SEAP Reporter Gene System
Luc Reporter Gene System
Ruc Reporter Gene System
Green Fluorescent Protein (GFP) Reporter Gene System
Bioluminescence Resonance Energy Transfer (BRET)
Human Growth Hormone (hGH) Reporter Gene System