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

DOI: 10.1186/1472-6750-8-81

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Abstract:

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.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.The zebrafish has been established as an excellent model for studying any biological process. This organism possesses many advantages including ease of experimentation, optical clarity, drug administration, amenability to in vivo manipulation and feasibility of reverse and forward genetic approaches. The fish reach sexual maturity in only 3 to 4 months, and adult females are capable of producing 100 to 200 eggs weekly. Many thousands of animals can be kept in a fish facility requiring much less space than mice or other mammals, and hence the zebrafish is regarded as a cost-effective experimental vertebrate model for large-scale genetic screening [1]. Furthermore, the high degree of homology between the zebrafish genome and that of humans makes such discoveries especially pertinent to human disease and development [2,3].Morpholino antisense oligonucleotides (MO) have been widely used to inhibit gene function in zebrafish embryos [4-7] and are usually used as sequence-specific translation-blo

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