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

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...

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Bibliographic Details
Published in:Mechanisms of development 2006-07, Vol.123 (7), p.513-529
Main Authors: Sivasubbu, Sridhar, Balciunas, Darius, Davidson, Ann E., Pickart, Michael A., Hermanson, Spencer B., Wangensteen, Kirk J., Wolbrink, Daniel C., Ekker, Stephen C.
Format: Article
Language:English
Subjects:
3′ gene-trap
Animals
Animals, Genetically Modified
DNA Transposable Elements - genetics
Gene-breaking
Histone H2afz
Histones - genetics
Histones - physiology
Insertional mutagenesis
Larva - genetics
Larva - growth & development
Mutagenesis, Insertional
Poly(A) trap
Sleeping Beauty
Transcription
Transposon
Zebrafish
Zebrafish - embryology
Zebrafish - genetics
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Summary: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.
ISSN:0925-4773
1872-6356
DOI:10.1016/j.mod.2006.06.002