Application of the Firefly Luciferase Reporter Gene

Abstract

Reporter genes have become powerful tools to study regulation of gene expression in eukaryotes. In particular, chimeric transcription units generated by the fusion of the appropriate DNA regulatory sequences to reporter genes have led to the identification of a great number of DNA control elements that constitute eukaryotic promoters. Recently, transcription factors that interact with specific DNA control elements have been purified and the cDNAs encoding these DNA-binding proteins have been cloned. Among these factors, the steroid hormone receptors have been shown to belong to a superfamily of transcription factors that regulate gene expression in a liganddependent fashion (1). The cloning of their respective cDNAs provided the opportunity to identifjr the functional domains for hormone binding, DNA binding, and transactivation present in these proteins. To perform these studies, we developed a screening assay that uses cultured cells transfected with two expression vectors (2). The first vector directs the expression of the wild-type or in vitro mutagenized receptor, and the second contains a reporter gene linked to a hormone-responsive promoter. Application of the hormone or a related synthetic drug activates the reporter gene, and the effects of the mutations on the ability of hormone/receptor complex to acti vate gene expression can be assessed. Because of the large number of experiments to be performed in these studies, the use of chloramphenicol acetyl transferase (CAT) (originally used in our cotransfection assay) as a reporter gene proved to be both laborious and onerous. In contrast, the luciferase gene possesses intrinsic qualities that most other reporter genes lack greater sensitivity, ease of use, instantaneous quantification, “ environment friendliness” (no radiolabeled compound or organic solvent is used in this assay), and cost efficiency. Here, the properties of the luciferase system are briefly discussed and a simple and rapid protocol used in my laboratory to assay luciferase activity in extract obtained from cultured mammalian cells transfected with a luciferase reporter gene.