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Open Access Highly Accessed Methodology article

Application of the dual-luciferase reporter assay to the analysis of promoter activity in Zebrafish embryos

Francisca Alcaraz-Pérez12, Victoriano Mulero2* and María L Cayuela1*

Author Affiliations

1 Research Unit, Department of Surgery, University Hospital "Virgen de la Arrixaca", 30120 Murcia, Spain

2 Department of Cell Biology and Histology, Faculty of Biology, University of Murcia, 30100 Murcia. Spain

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BMC Biotechnology 2008, 8:81  doi:10.1186/1472-6750-8-81

Published: 27 October 2008

Abstract

Background

The dual-luciferase assay has been widely used in cell lines to determine rapidly but accurately the activity of a given promoter. Although this strategy has proved very useful, it does not allow the promoter and gene function to be analyzed in the context of the whole organism.

Results

Here, we present a rapid and sensitive assay based on the classical dual-luciferase reporter technique which can be used as a new tool to characterize the minimum promoter region of a gene as well as the in vivo response of inducible promoters to different stimuli. We illustrate the usefulness of this system for studying both constitutive (telomerase) and inducible (NF-κB-dependent) promoters. The flexibility of this assay is demonstrated by induction of the NF-κB-dependent promoters using simultaneous microinjection of different pathogen-associated molecular patterns as well as with the use of morpholino-gene mediated knockdown.

Conclusion

This assay has several advantages compared with the classical in vitro (cell lines) and in vivo (transgenic mice) approaches. Among others, the assay allows a rapid and quantitative measurement of the effects of particular genes or drugs in a given promoter in the context of a whole organism and it can also be used in high throughput screening experiments.