Thank you for visiting nature. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.
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.
A single person can prepare a dozen constructs within 3 weeks, and obtain transgenic fish within approximately 3β4 months. Our protocol drastically reduces the labor involved in BAC transgenesis and will greatly facilitate biological and biomedical studies in model vertebrates. BAC transgenesis has been used extensively in a broad range of applications in mice, from studies of gene regulation to creating animal models of human disease 1 , 2 , 3. Because BACs can hold genomic fragments as large as kb, they often include the complete structure of a gene, including long-range cis -regulatory elements required for correct cell typeβspecific and temporal expression.
Compared with small plasmid-based transgenes, BACs are generally more resistant to positional effects, presumably because of their larger size. BAC transgenic mice have uniquely advanced the analysis of distant cis -regulatory elements, rescue of mutant phenotypes and study of human disease-related genes 1 , 2. BAC reporter transgenes have been created systematically by recombineering fluorescent reporters in bacteria to visualize specific tissues or cells in living mice 4 , 5.
Given the tractability of zebrafish Danio rerio for vertebrate genetics, improving the frequency and reliability of BAC transgenesis in this model organism could have a number of important advantages. BAC transgenic zebrafish have been reported for more than 10 years 6 , 7 , 8 ; however, systematic generation of BAC reporter lines has not been straightforward.
