Transposon-mediated BAC transgenesis in zebrafish

Maximiliano L. Suster, Gembu Abe, Anders Schouw, Koichi Kawakami

Research output: Contribution to journalArticle

117 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)1998-2021
Number of pages24
JournalNature Protocols
Volume6
Issue number12
DOIs
Publication statusPublished - 2011 Dec 1
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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