Seeing the Light: Luminescent Reporter Gene Assays

Loren      J. Miraglia; Frederick      J. King; Robert      Damoiseaux

Abstract

The luminescent reporter gene assay (LRGA) is arguably the most prominent type of reporter gene assay used in biomolecular and pharmaceutical development laboratories. Part of this popularity is due to the high signal associated with luciferases, the foundation of this technology. This feature makes them ideally suited for high throughput screening applications where potentially millions of chemical compounds can be analyzed in a given assay. Recent technical advancements that enhance signal stability of the luciferases along with development and commercialization of multiple forms of luciferases, their respective substrates, and improvements in expression vectors for reporter gene assay (RGA) applications have broadened their use. While the practical challenges related to the application of luminescent technology in a laboratory setting have been overcome, there remains much to do in laying a systematic approach towards the construction of RGAs, which are essential to the elucidation of the basic biology for genes of interest. This mini-review aims at giving a birds-eye view of the available luciferases, substrates and other luminescent technologies available and provides a general blueprint as well as practical considerations for constructing and interfacing RGAs with chemical biology and functional genomics for the elucidation of fundamental biological questions and for biomedical research.

Keywords: Luciferase Renilla, firefly, reporter gene assay (RGA), promoter, luciferin, high throughput screening, biomedical research, irradiation, Coelenterazine, co-enzyme, promoters, TATA box, cDNA