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Abstract
BackgroundExternal 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.ResultsWe 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: nsf tpl6, atp1a3atpl10 and flrtpl19. Our gene trap cassette is flanked by direct loxP sites, which enabled us to successfully revert nsf tpl6, 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.ConclusionsThe 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.
Highlights
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
We provide proof-of-principle demonstration that a gene breaking transposon can be equipped with Gal4-VP16, resulting in sensitive detection of weak gene expression
Gene trap vector design and features Our Gene Breaking Transposon (GBT)-B1 vector is composed of several components that in concert ensure efficient mutagenesis, evaluation of the trapped gene’s expression profile and enable manipulation of resulting mutant alleles
Summary
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. Two large-scale ethylnitrosourea (ENU) mutagenesis screens have clearly established that all aspects of zebrafish development can be analyzed by forward genetics [2,3,4]. Insertional mutagenesis is not as random as chemical mutagenesis and does not possess as high efficiency These deficiencies are offset by more straightforward identification of affected genes using the insertional mutagen as a molecular tag. Production and handling of viral particles requires special expertise and facilities Modifications such as addition of gene trap components may result in lower virus titers and require significant optimization of production procedures [15]. The only retroviral approach that produced fluorescent protein expression-tagged integration events -the enhancer trap- did not produce a significant number of loss-of-function alleles [14]
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