title: Gene-breaking transposon mutagenesis reveals an essential role for histone H2afza in zebrafish larval development.
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.
authors.affiliation: University 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.
authors.forename: Sridhar
authors.initials: S
authors.lastname: Sivasubbu
authors.forename: Darius
authors.initials: D
authors.lastname: Balciunas
authors.forename: Ann E
authors.initials: AE
authors.lastname: Davidson
authors.forename: Michael A
authors.initials: MA
authors.lastname: Pickart
authors.forename: Spencer B
authors.initials: SB
authors.lastname: Hermanson
authors.forename: Kirk J
authors.initials: KJ
authors.lastname: Wangensteen
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Wolbrink
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Ekker
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D004251:DNA Transposable Elements / Q000235:genetics*; D006657:Histones / Q000235:genetics* / Q000502:physiology; D007814:Larva / Q000235:genetics / Q000254:growth & development; D016254:Mutagenesis, Insertional; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*
chemical_list: D004251:DNA Transposable Elements; D006657:Histones; C077020:histone H2A.F-Z