Reporter gene assays are a laboratory technique used to measure gene activity. The more commonly used reporter gene assays include:
Green Fluorescent Protein (GFP) emits a green glow under a UV light. GFP assays are easy to perform and visualize using fluorescence microscopy and are widely used for studying gene regulation and quantifying gene expression. Flow cytometry is used to count individual cells such as when monitoring transfection efficiency.
Luciferase, often derived from fireflies, produces blue or yellow-green light when it reacts with a specific substrate. Luciferase assays are highly sensitive and have a wide dynamic range, allowing detection of even tiny changes in gene activity. Luciferase bioluminescence does not require light excitation, resulting in minimum auto fluorescence, which allows the fluorescence signal to be detected virtually background-free. Similar to GFP, luciferase is widely used for studying gene expression and regulation. Additionally, different types of luciferase emit different colored light, allowing scientists to study multiple genes simultaneously.
β-glucuronidase (GUS) comes with multiple assay formats. A widely used format involves staining cells using a histochemical technique. When the GUS gene is activated, it turns a colorless or non-fluorescent solution into a colored or fluorescent product, making it easy to see under a microscope. The GUS assay offers flexibility with different detection methods and is particularly useful for studying gene regulation in plants. However, a downside is that the staining process kills the cells.
β-galactosidase (LacZ), which encodes a β-galactosidase enzyme, breaks down a colorless substrate into a blue product, allowing for easy detection with the naked eye. β-galactosidase assays are widely used for identifying bacteria that are transformed and harboring recombinant plasmids. The lacZ gene activity is interrupted when a foreign DNA is inserted into the multiple cloning site of an expression plasmid. This disrupts production of β-galactosidase, causing the recombinant bacterial colonies to appear white in color. Non-recombinant colonies, on the other hand, appear blue in color. This simple color change makes this a popular assay in cloning experiments. However, its sensitivity is lower than luciferase assays.