In Vitro Reporter Assays for Screening of Chemicals That Disrupt Androgen Signaling
Table 2
Commonly used reporter genes and their characteristics.
S. number
Reporter gene
Function
Advantages
Disadvantages
1
-Galactosidase (lacZ)
First reported in 1980. In E. coli, hydrolyses lactose to glucose and galactose.
Can act on many substrates.
Costly and potentially toxic chemical for assay and lysis of cells. Not useful for real time detection systems.
2
Luciferase (eukaryotic or bacterial)
Proteins that generate luminescence biologically. Can be eukaryotic or bacterial (lux). Firefly luciferase is one of the most common reporter genes.
High sensitivity, tight coupling of Luc protein with luminescence output, protein requires no posttranslational modification.
Firefly luciferase requires addition of costly substrate luciferin to monitor activity. The substrate for bacterial luciferase is produced endogenously, but not very active in eukaryotic systems.
3
GFP
Originally isolated from Jelly fish Aequorea victoria; gene was cloned later.
Functional in both prokaryotic and eukaryotic systems. Broad host applicability in absence of cell lysis or substrate addition.
These stable proteins continue to emit fluorescence long after the host has died. The fluorophore within wild type GFP needs two hours for generation.
4
CAT
The enzyme is found in prokaryotes. Transfers acetyl group from acetyl coA molecule to chloramphenicol, causing its detoxification.
Gene product is stable and detectable at attomolar concentrations. Suitable for mammalian systems.
