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.
Original language | English (US) |
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Pages (from-to) | 513-529 |
Number of pages | 17 |
Journal | Mechanisms of Development |
Volume | 123 |
Issue number | 7 |
DOIs | |
State | Published - Jul 2006 |
Keywords
- 3′ gene-trap
- Gene-breaking
- Histone H2afz
- Insertional mutagenesis
- Poly(A) trap
- Sleeping Beauty
- Transcription
- Transposon
- Zebrafish
ASJC Scopus subject areas
- Embryology
- Developmental Biology