General Description
Analysis of gene expression is commonly assayed by transient transfection. These systems are usually based on the use of fusion genes which are inserted into cells, and expression of the gene is assayed within 48 hours after introduction of DNA. Usually the fusion consists of the promoter binding site or enhancer sequence under study which is attached to a reporter gene. The amount of the reporter protein synthesized under the experimental conditions is presumed to reflect the ability of the sequences studied to direct or promote transcription.
Luciferase has become one of the more widely used reporter enzymes. The reporter plasmid contains the gene from the firefly Photinus pyralis. The enzyme catalyzes a bioluminescent reaction which requires the substrate luciferin as well as Mg
+2 and ATP. Mixing these reagents with the cell extract containing luciferase results in a flash of light that decays rapidly. This light can be detected by a luminometer. The total light emission is proportional to the luciferase activity of the sample. The luciferase assay is fast and sensitive and does not require a radioactive substrate as in the CAT assay. A disadvantage of the luciferase assay is that it requires a rather expensive instrument, the luminometer, to measure enzyme activity.
