Journal
NATURE PROTOCOLS
Volume 6, Issue 12, Pages 1998-2021Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2011.416
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Funding
- Sars International Centre
- Japan Society for the Promotion of Science
- Takeda Science Foundation
- National BioResource Project of Japan
- Ministry of Education, Culture, Sports, Science and Technnology of Japan
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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 similar to 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.
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