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The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach
Published on: November 30, 2022
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A Plasmid Set for Efficient Bacterial Artificial Chromosome (BAC) Transgenesis in Zebrafish
Fernando Fuentes1, Eric Reynolds1, Stephen W Lewellis1
1Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, New York University Langone Medical Center, New York 10016.
G3 (Bethesda, Md.)
|January 29, 2016
Summary
Tol2 transposase facilitates large DNA construct transgenesis in zebrafish using bacterial artificial chromosomes (BACs). This method enables efficient gene function analysis by accurately reflecting endogenous gene expression patterns.
Area of Science:
- Genetics
- Molecular Biology
- Developmental Biology
Background:
- Transgenesis of large DNA constructs is crucial for understanding gene function.
- Tol2 transposase-mediated transgenesis is a powerful method for inserting bacterial artificial chromosome (BAC) DNA into zebrafish genomes.
- Efficient transgenesis requires Tol2 transposon sites flanking the DNA and a transgenesis marker for identification.
Purpose of the Study:
- To develop and validate plasmids for inserting Tol2 sites and transgenesis markers into BACs.
- To assess the efficiency of Tol2 transposase-mediated BAC transgenesis in zebrafish.
- To evaluate the utility of BAC transgenics for recapitulating endogenous gene expression.
Main Methods:
- Development of targeting cassettes for BAC modification.
- Tol2 transposase-mediated insertion of modified BACs into zebrafish.
- Selection of transgenic zebrafish based on transgenesis marker expression.
- Analysis of gene expression patterns in BAC transgenics compared to endogenous levels.
Main Results:
- The developed plasmids efficiently insert Tol2 sites and markers into BACs.
- Tol2 transposase-mediated BAC transgenesis efficiency is comparable to existing methods (iTol2).
- Preselection for marker expression significantly increases transgenesis rates.
- BAC transgenics accurately reflect endogenous gene expression patterns and levels.
Conclusions:
- The described plasmid system provides an efficient method for Tol2 transposase-mediated BAC transgenesis in zebrafish.
- This approach facilitates robust gene function analysis by enabling faithful recapitulation of endogenous gene expression.
- The method allows for accurate estimation of endogenous gene expression levels using BAC transgenics.
