title: Transposon-mediated BAC transgenesis in zebrafish.
abstract: Bacterial artificial chromosomes (BACs) are widely used in studies of vertebrate gene regulation and function because they often closely recapitulate the expression patterns of endogenous genes. Here we report a step-by-step protocol for efficient BAC transgenesis in zebrafish using the medaka Tol2 transposon. Using recombineering in Escherichia coli, we introduce the iTol2 cassette in the BAC plasmid backbone, which contains the inverted minimal cis-sequences required for Tol2 transposition, and a reporter gene to replace a target locus in the BAC. Microinjection of the Tol2-BAC and a codon-optimized transposase mRNA into fertilized eggs results in clean integrations in the genome and transmission to the germline at a rate of ∼15%. A single person can prepare a dozen constructs within 3 weeks, and obtain transgenic fish within approximately 3-4 months. Our protocol drastically reduces the labor involved in BAC transgenesis and will greatly facilitate biological and biomedical studies in model vertebrates.
authors: Division of Molecular and Developmental Biology, National Institute of Genetics, Mishima, Shizuoka, Japan. maximiliano.suster@sars.uib.no
authors: Maximiliano L
authors: ML
authors: Suster
authors: Gembu
authors: G
authors: Abe
authors: Anders
authors: A
authors: Schouw
authors: Koichi
authors: K
authors: Kawakami
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics; D018014:Gene Transfer Techniques; D011995:Recombination, Genetic; D015027:Zebrafish / Q000235:genetics*
chemical_list: D004251:DNA Transposable Elements