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The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach
Published on: November 30, 2022
Transposon-mediated BAC transgenesis in zebrafish
Maximiliano L Suster1, Gembu Abe, Anders Schouw
1Division of Molecular and Developmental Biology, National Institute of Genetics, Mishima, Shizuoka, Japan. maximiliano.suster@sars.uib.no
Nature Protocols
|December 3, 2011
Summary
This study presents an efficient protocol for creating bacterial artificial chromosome (BAC) transgenic zebrafish using the Tol2 transposon system. This method simplifies BAC transgenesis, accelerating vertebrate gene studies.
Area of Science:
- Molecular Biology
- Genetics
- Developmental Biology
Background:
- Bacterial artificial chromosomes (BACs) are crucial for studying vertebrate gene regulation due to their ability to mimic endogenous gene expression.
- Efficient BAC transgenesis is essential for advancing research in model vertebrates.
Purpose of the Study:
- To develop and present a streamlined, step-by-step protocol for efficient BAC transgenesis in zebrafish.
- To utilize the medaka Tol2 transposon system for enhanced BAC integration and germline transmission.
Main Methods:
- Employing recombineering in Escherichia coli to engineer BAC plasmids with the iTol2 cassette and reporter genes.
- Introducing the inverted minimal cis-sequences for Tol2 transposition and replacing target loci within the BAC.
- Microinjecting the engineered Tol2-BAC construct and codon-optimized transposase mRNA into zebrafish embryos.
Main Results:
- Achieved clean genomic integrations of BAC constructs in zebrafish.
- Demonstrated germline transmission of the transgenes at a rate of approximately 15%.
- Established a protocol enabling a single researcher to prepare multiple constructs within 3 weeks and obtain transgenic fish in 3-4 months.
Conclusions:
- The developed protocol significantly reduces labor and time for BAC transgenesis in zebrafish.
- This efficient method will greatly facilitate biological and biomedical research involving model vertebrates.
- The Tol2 transposon-based BAC system offers a powerful tool for gene regulation and function studies.
