Transposon-mediated BAC transgenesis in zebrafish
Maximiliano L Suster, Gembu Abe, Anders Schouw and Koichi Kawakami
Nature Protocols Vol 6, 1998-2021 Year: 2011
DOI: 10.1038/nprot.2011.416
Bacterial artificial chromosomes (BACs) are widely used in studies of vertebrate gene regulation and function because they closely recapitulate the expression patterns of endogenous genes. BAC DNA has been introduced into the zebrafish genome by microinjection of the naked BAC clones into fertilized eggs, but the frequency of recovery of transgenic offspring is low. Recently, we developed efficient BAC transgenesis method in zebrafish using the medaka Tol2 transposon. Using recombineering technique in Escherichia coli, the iTol2 cassette, which contains the minimal cis-sequences required for Tol2 transposition, is introduced into a BAC plasmid. Microinjection of the Tol2-BAC and the transposase mRNA into fertilized eggs results in clean integrations in the genome and transmission to the next generation at a rate of ~15%. A single person can prepare a dozen of constructs within 3 weeks, and obtain transgenic fish within approximately 3-4 months. Our protocol drastically reduces the labor required for BAC transgenesis and will greatly facilitate biological and biomedical studies in model vertebrates.
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A scheme for Tol2 transposon-mediated BAC transgenesis in zebrafish. Tol2-BAC construct containing the Tol2 sequences (red triangle in a box) is injected with transposase mRNA into fertilized eggs, and is integrated into the zebrafish genome efficiently.
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