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Open Access Methodology article

Efficient disruption of Zebrafish genes using a Gal4-containing gene trap

Jorune Balciuniene1, Danielle Nagelberg1, Kathleen T Walsh1, Diana Camerota1, Daphné Georlette1, Frédéric Biemar1, Gianfranco Bellipanni12 and Darius Balciunas1*

Author Affiliations

1 Department of Biology, College of Science and Technology, Temple University, Philadelphia, PA 19122, USA

2 Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, Philadelphia, PA 19122, USA

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BMC Genomics 2013, 14:619  doi:10.1186/1471-2164-14-619

Published: 14 September 2013

Abstract

Background

External development and optical transparency of embryos make zebrafish exceptionally suitable for in vivo insertional mutagenesis using fluorescent proteins to visualize expression patterns of mutated genes. Recently developed Gene Breaking Transposon (GBT) vectors greatly improve the fidelity and mutagenicity of transposon-based gene trap vectors.

Results

We constructed and tested a bipartite GBT vector with Gal4-VP16 as the primary gene trap reporter. Our vector also contains a UAS:eGFP cassette for direct detection of gene trap events by fluorescence. To confirm gene trap events, we generated a UAS:mRFP tester line. We screened 270 potential founders and established 41 gene trap lines. Three of our gene trap alleles display homozygous lethal phenotypes ranging from embryonic to late larval: nsftpl6, atp1a3atpl10 and flrtpl19. Our gene trap cassette is flanked by direct loxP sites, which enabled us to successfully revert nsftpl6, atp1a3atpl10 and flrtpl19 gene trap alleles by injection of Cre mRNA. The UAS:eGFP cassette is flanked by direct FRT sites. It can be readily removed by injection of Flp mRNA for use of our gene trap alleles with other tissue-specific GFP-marked lines. The Gal4-VP16 component of our vector provides two important advantages over other GBT vectors. The first is increased sensitivity, which enabled us to detect previously unnoticed expression of nsf in the pancreas. The second advantage is that all our gene trap lines, including integrations into non-essential genes, can be used as highly specific Gal4 drivers for expression of other transgenes under the control of Gal4 UAS.

Conclusions

The Gal4-containing bipartite Gene Breaking Transposon vector presented here retains high specificity for integrations into genes, high mutagenicity and revertibility by Cre. These features, together with utility as highly specific Gal4 drivers, make gene trap mutants presented here especially useful to the research community.

Keywords:
Zebrafish; Insertional mutagenesis; Gene trap; Gal4; Tol2; nsfa; Fleer; atp1a3a; bbs7