Efficient activation of gene expression using a heat-shock inducible Gal4/Vp16-UAS system in medaka

Clemens Grabher,Joachim Wittbrodt

doi:10.1186/1472-6750-4-26

Clemens Grabher, Joachim Wittbrodt

Open Access

https://doi.org/10.1186/1472-6750-4-26

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Journal: BMC biotechnology	Publication Date: Jan 1, 2004
Citations: 55	License type: cc-by

Affiliation: European Molecular Biology Laboratory

Abstract

BackgroundGenetic interference by DNA, mRNA or morpholino injection is a widely used approach to study gene function in developmental biology. However, the lack of temporal control over the activity of interfering molecules often hampers investigation of gene function required during later stages of embryogenesis. To elucidate the roles of genes during embryogenesis a precise temporal control of transgene expression levels in the developing organism is on demand.ResultsWe have generated a transgenic Gal4/Vp16 activator line that is heat-shock inducible, thereby providing a tool to drive the expression of specific effector genes via Gal4/Vp16. Merging the Gal4/Vp16-UAS system with the I-SceI meganuclease and the Sleeping Beauty transposon system allows inducible gene expression in an entirely uniform manner without the need to generate transgenic effector lines. Combination of this system with fluorescent protein reporters furthermore facilitates the direct visualization of transgene expressing cells in live embryos.ConclusionThe combinatorial properties of this expression system provide a powerful tool for the analysis of gene function during embryonic and larval development in fish by ectopic expression of gene products.

Highlights

Genetic interference by DNA, mRNA or morpholino injection is a widely used approach to study gene function in developmental biology
Generation of a heat-shock inducible transgenic Gal4/ Vp16 activator line DNA injection leading to mosaic expression in G0 allows in vivo tracing of transgene-expressing cells and observation of effects exerted by the transgene through application of fluorescent markers [11]
While the MN protocol strongly reduces mosaicism, it does so only in a fraction of injected embryos ([13], Fig. 1F,1G,1H,1I,1J,1K and Table 1). This can be improved by the use of transgenic animals providing inducible and sufficient expression in all cells

Summary

Introduction

Genetic interference by DNA, mRNA or morpholino injection is a widely used approach to study gene function in developmental biology. The lack of temporal control over the activity of interfering molecules often hampers investigation of gene function required during later stages of embryogenesis. To elucidate the roles of genes during embryogenesis a precise temporal control of transgene expression levels in the developing organism is on demand. MRNA and morpholinos exert their functions immediately following injection, providing information only on the early role of the gene of interest. The generation of different transgenic activator and effector lines may be a time- and space-consuming task, and expression levels in these transgenic lines are weak, probably due to a limited transactivation potential of Gal in fish. Strong transcriptional activators can cause unspecific promoter squelching [9] resulting in retardation of embryogenesis [10]

Methods

Results

Conclusion