title: A Plasmid Set for Efficient Bacterial Artificial Chromosome (BAC) Transgenesis in Zebrafish.
abstract: Transgenesis of large DNA constructs is essential for gene function analysis. Recently, Tol2 transposase-mediated transgenesis has emerged as a powerful tool to insert bacterial artificial chromosome (BAC) DNA constructs into the genome of zebrafish. For efficient transgenesis, the genomic DNA piece in the BAC construct needs to be flanked by Tol2 transposon sites, and the constructs should contain a transgenesis marker for easy identification of transgenic animals. We report a set of plasmids that contain targeting cassettes that allow the insertion of Tol2 sites and different transgenesis markers into BACs. Using BACs containing these targeting cassettes, we show that transgenesis is as efficient as iTol2, that preselecting for expression of the transgenesis marker increases the transgenesis rate, and that BAC transgenics faithfully recapitulate the endogenous gene expression patterns and allow for the estimation of the endogenous gene expression levels.
authors.affiliation: Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, New York University Langone Medical Center, New York 10016.
authors.forename: Fernando
authors.initials: F
authors.lastname: Fuentes
authors.affiliation: Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, New York University Langone Medical Center, New York 10016.
authors.forename: Eric
authors.initials: E
authors.lastname: Reynolds
authors.affiliation: Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, New York University Langone Medical Center, New York 10016.
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Lewellis
authors.affiliation: Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, New York University Langone Medical Center, New York 10016.
authors.forename: Gayatri
authors.initials: G
authors.lastname: Venkiteswaran
authors.affiliation: Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, New York University Langone Medical Center, New York 10016 Kimmel Center for Stem Cell Biology, New York University Langone Medical Center, New York 10016 holger.knaut@med.nyu.edu.
authors.forename: Holger
authors.initials: H
authors.lastname: Knaut
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D022202:Chromosomes, Artificial, Bacterial; D004625:Embryo, Nonmammalian / Q000378:metabolism; D015870:Gene Expression; D023061:Gene Order; D018390:Gene Targeting; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D019076:Transgenes; D015027:Zebrafish / Q000235:genetics*
keywords: BAC transgenesis; gene expression; zebrafish
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