ZFIN ID: ZDB-PUB-060731-2
Gene-breaking transposon mutagenesis reveals an essential role for histone H2afza in zebrafish larval development
Sivasubbu, S., Balciunas, D., Davidson, A.E., Pickart, M.A., Hermanson, S.B., Wangensteen, K.J., Wolbrink, D.C., and Ekker, S.C.
Date: 2006
Source: Mechanisms of Development 123(7): 513-529 (Journal)
Registered Authors: Balciunas, Darius, Ekker, Stephen C., Hermanson, Spencer, Pickart, Michael, Sivasubbu, Sridhar
Keywords: Insertional mutagenesis, Poly(A) trap, 32 gene-trap, Gene-breaking, Sleeping Beauty, Transposon, Zebrafish, Transcription, Histone H2afz
MeSH Terms: Animals; Animals, Genetically Modified; DNA Transposable Elements/genetics*; Histones/genetics*; Histones/physiology (all 10) expand
PubMed: 16859902 Full text @ Mech. Dev.
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.
ADDITIONAL INFORMATION