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Mech Dev. 2006 Jul;123(7):513-29. Epub 2006 Jun 9.

Gene-breaking transposon mutagenesis reveals an essential role for histone H2afza in zebrafish larval development.

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  • 1University of Minnesota, Department of Genetics, Cell Biology and Development, Arnold and Mabel Beckman Center for Transposon Research, 321 Church St SE, 6-160 Jackson Hall, Minneapolis, MN 55455, USA.

Abstract

We report a novel gene tagging, identification and mutagenicity ('gene-breaking') method for the zebrafish, Danio rerio. This modular approach consists of two distinct and separable molecular cassettes. The first is a gene-finding cassette. In this study, we employed a 3' gene-tagging approach that selectively 'traps' transcripts regardless of expression status, and we show that this cassette identifies both known and novel endogenous transcripts in transgenic zebrafish. The second is a transcriptional termination mutagenicity cassette assembled from a combination of a splice acceptor and polyadenylation signal to disrupt tagged transcripts upon integration into intronic sequence. We identified both novel and conserved loci as linked phenotypic mutations using this gene-breaking strategy, generating molecularly null mutations in both larval lethal and adult viable loci. We show that the Histone 2a family member z (H2afza) variant is essential for larval development through the generation of a lethal locus with a truncation of conserved carboxy-terminal residues in the protein. In principle this gene-breaking strategy is scalable for functional genomics screens and can be used in Sleeping Beauty transposon and other gene delivery systems in the zebrafish.

PMID:
16859902
[PubMed - indexed for MEDLINE]
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