Secreted dual reporter assay with Gaussia luciferase and the red fluorescent protein mCherry.

Diana Wider,Didier Picard

doi:10.1371/journal.pone.0189403

Abstract

The availability of a wide range of reporter proteins, which can easily be quantitated, has had a major impact on many fields of biomedical research. In some experiments with tissue culture cells, it is necessary to control for differences in transfection efficiency and in other expression parameters. This requirement has been very conveniently met with the popular dual luciferase assay. Its disadvantages are the requirement for cell lysis, the inability to analyze the same cells repeatedly, and the cost, at least in its most commonly used commercial format. Here we describe a novel dual reporter assay with the naturally secreted luciferase from Gaussia princeps as the main reporter protein and a secreted version of the red fluorescent protein mCherry as internal standard. After first measuring mCherry fluorescence in the medium, an enzyme buffer with coelenterazine as substrate is added to the same sample to trigger a glow-type luminescence of the luciferase. The simple and cheap assay can easily be adapted to a variety of experimental situations. As a case in point, we have developed a panel of Gaussia luciferase reporter genes for transcriptional activation assays with estrogen and glucocorticoid response elements, and with response elements for fusion proteins with the Gal4 DNA binding domain for use in mammalian cells. Our secreted dual reporter assay should be an attractive alternative to the currently available commercial kits.

Highlights

The development of reporter genes has enormously facilitated the analysis of many steps of gene expression, from signal transduction to gene regulation to translation and protein trafficking and targeting
The well-known dual luciferase assay consists of the consecutive enzymatic analysis of the activities of the Photinus pyralis ("firefly") and Renilla reniformis ("Renilla") intracellular luciferases
Our efforts were directed at mammalian tissue culture cells, but the system should be applicable to tissue culture cells of other species as well

Summary

Introduction

The development of reporter genes has enormously facilitated the analysis of many steps of gene expression, from signal transduction to gene regulation to translation and protein trafficking and targeting. Chloramphenicol acetyltransferase, known as CAT, was one of the first to be introduced as a reporter enzyme for transcriptional assays in tissue culture cells [1]. The luciferase of the firefly Photinus pyralis was cloned and became a serious competitor for CAT because of the simplicity of the assay [2,3,4,5]. By the early 1990s, there were a whole panel of different enzymatic, including bioluminescent, and chemiluminescent reporter proteins [6,7]. In those days, firefly luciferase was an extremely convenient reporter protein, it was not trivial to standardize its activities in transfection experiments with tissue.

Methods

Results

Conclusion