PUBLICATION

Transposon-mediated BAC transgenesis in zebrafish

Authors
Suster, M.L., Abe, G., Schouw, A., and Kawakami, K.
ID
ZDB-PUB-111205-13
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.
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.

Genes / Markers
Figures
Expression
Phenotype
Mutation and Transgenics
Human Disease / Model Data
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping
Errata and Notes