Transposon-mediated BAC transgenesis in zebrafish

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

研究成果: Article

119 引用 (Scopus)

抜粋

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.

元の言語English
ページ(範囲)1998-2021
ページ数24
ジャーナルNature Protocols
6
発行部数12
DOI
出版物ステータスPublished - 2011 12 1
外部発表Yes

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

フィンガープリント Transposon-mediated BAC transgenesis in zebrafish' の研究トピックを掘り下げます。これらはともに一意のフィンガープリントを構成します。

  • これを引用