Transposon-mediated BAC transgenesis in zebrafish and mice

We took advantage of the precise cut-and-paste behavior of a natural transposon, Tol2, to develop a new method for BAC transgenesis. In this new method, the minimal sequences of the Tol2 transposon were used to deliver precisely single copies of a ~70 kb BAC transgene to the zebrafish and mouse genomes. We mapped the BAC insertion sites in the genome by standard PCR methods and confirmed transposase-mediated integrations.The Tol2 transposon has a surprisingly large cargo capacity that can be harnessed for BAC transgenesis. The precise delivery of single-copy BAC transgenes by Tol2 represents a useful complement to conventional BAC transgenesis, and could aid greatly in the production of transgenic fish and mice for genomics projects, especially those in which single-copy integrations are desired.BAC transgenesis has many important applications in basic and applied biomedical research, from the analysis of regulatory elements controlling gene expression to creating animal models of human diseases [1-3]. Because BACs can hold genomic segments as large as 300 kb, they are invaluable for the analysis of distant cis-regulatory elements [4], rescue of mutant phenotypes and regulated expression of human disease-related genes in mice [2,3]. A number of BACs encoding fluorescent reporters have been created by 'recombineering' in bacteria to visualize specific tissues or cell types in vivo [4-6].BAC transgenesis has been carried out by microinjection of naked DNA into the cytoplasm of fertilized eggs in zebrafish [4,5] or oocyte pronucleus in mice [2,3]. With these methods, integration of the BAC in the genome occurs at best in ~5-20% of the injected survivors in mice [3] and ~1-3% in zebrafish [5]. A recent extensive study showed that about half of BAC transgenic mice carried 1 to 5 copies of the injected BAC as concatamers at one genomic locus, and the rest carried more than 5 copies, and up to 48 copies, arranged in various orientations [7]. Concatameric transgenes may be