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ZFIN ID: ZDB-PUB-111205-13
Transposon-mediated BAC transgenesis in zebrafish
Suster, M.L., Abe, G., Schouw, A., and Kawakami, K.
Date: 2011
Source: Nature Protocols   6(12): 1998-2021 (Journal)
Registered Authors: Kawakami, Koichi, Suster, Maximiliano
Keywords: developmental biology, genetic analysis, genetic modification, genomics, model organisms
MeSH Terms:
  • Animals
  • Animals, Genetically Modified
  • Chromosomes, Artificial, Bacterial/genetics*
  • DNA Transposable Elements*
  • Escherichia coli/genetics
  • Gene Transfer Techniques*
  • Recombination, Genetic
  • Zebrafish/genetics*
PubMed: 22134125 Full text @ Nat. Protoc.

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.