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

Sridhar Sivasubbu; Darius Balciunas; Ann E. Davidson; Michael A. Pickart; Spencer B. Hermanson; Kirk J. Wangensteen; Daniel C. Wolbrink; Stephen C. Ekker

doi:10.1016/j.mod.2006.06.002

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

Sridhar Sivasubbu, Darius Balciunas, Ann E. Davidson, Michael A. Pickart, Spencer B. Hermanson, Kirk J. Wangensteen, Daniel C. Wolbrink, Stephen C. Ekker

Biochemistry and Molecular Biology

Research output: Contribution to journal › Article › peer-review

84 Scopus citations

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)
Pages (from-to)	513-529
Number of pages	17
Journal	Mechanisms of Development
Volume	123
Issue number	7
DOIs	https://doi.org/10.1016/j.mod.2006.06.002
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

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Gene-breaking transposon mutagenesis reveals an essential role for histone H2afza in zebrafish larval development. / Sivasubbu, Sridhar; Balciunas, Darius; Davidson, Ann E.; Pickart, Michael A.; Hermanson, Spencer B.; Wangensteen, Kirk J.; Wolbrink, Daniel C.; Ekker, Stephen C.

In: Mechanisms of Development, Vol. 123, No. 7, 07.2006, p. 513-529.

Research output: Contribution to journal › Article › peer-review

@article{01eaeb5fe0664187a68bac7442b17c6b,

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.",

keywords = "3′ gene-trap, Gene-breaking, Histone H2afz, Insertional mutagenesis, Poly(A) trap, Sleeping Beauty, Transcription, Transposon, Zebrafish",

author = "Sridhar Sivasubbu and Darius Balciunas and Davidson, {Ann E.} and Pickart, {Michael A.} and Hermanson, {Spencer B.} and Wangensteen, {Kirk J.} and Wolbrink, {Daniel C.} and Ekker, {Stephen C.}",

note = "Funding Information: We thank Amber Genetzky, Aubrey Nielsen and Brent Bill for help with fish screening, and Rachel Bowers and Dan Carlson for fish maintenance and Jon Larson for help with in situ hybridization. We thank Anthony Person and Jon Larson for comments on the manuscript. We also thank all members of the Arnold and Mabel Beckman Center for Transposon Research for valuable discussion. Research reagents described in this study are readily available to the scientific community and can be obtained by contacting the authors or visiting the Arnold and Mabel Beckman Center for Transposon Research website at http://beckmancenter.ahc.umn.edu . The research was supported by the Arnold and Mabel Beckman Foundation and the National Institutes of Health (DA14546).",

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AU - Balciunas, Darius

AU - Davidson, Ann E.

AU - Pickart, Michael A.

AU - Hermanson, Spencer B.

AU - Wangensteen, Kirk J.

AU - Wolbrink, Daniel C.

AU - Ekker, Stephen C.

N1 - Funding Information: We thank Amber Genetzky, Aubrey Nielsen and Brent Bill for help with fish screening, and Rachel Bowers and Dan Carlson for fish maintenance and Jon Larson for help with in situ hybridization. We thank Anthony Person and Jon Larson for comments on the manuscript. We also thank all members of the Arnold and Mabel Beckman Center for Transposon Research for valuable discussion. Research reagents described in this study are readily available to the scientific community and can be obtained by contacting the authors or visiting the Arnold and Mabel Beckman Center for Transposon Research website at http://beckmancenter.ahc.umn.edu . The research was supported by the Arnold and Mabel Beckman Foundation and the National Institutes of Health (DA14546).

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N2 - 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.

AB - 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.

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