Evaluating the biological relevance of putative enhancers using Tol2 transposon-mediated transgenesis in zebrafish

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Abstract
Evaluating the biological relevance of the myriad putative regulatory noncoding sequences in vertebrate genomes represents a huge challenge. Functional analyses in vivo have typically relied on costly and labor-intensive transgenic strategies in mice. Transgenesis has also been applied in nonrodent vertebrates, such as zebrafish, but until recently these efforts have been hampered by significant mosaicism and poor rates of germline transmission. We have developed a transgenic strategy in zebrafish based on the Tol2 transposon, a mobile element that was recently identified in another teleost, Medaka. This method takes advantage of the increased efficiency of genome integration that is afforded by this intact DNA transposon, activity that is mediated by the corresponding transposase protein. The approach described in this protocol uses a universal vector system that permits rapid incorporation of DNA that is tagged with sequence targets for site-specific recombination. To evaluate the regulatory potential of a candidate sequence, the desired interval is PCR-amplified using sequence-specific primers that are flanked by the requisite target sites for cloning, and recombined into a universal expression plasmid (pGW_cfosEGFP). Purified recombinant DNAs are then injected into 1-2-cell zebrafish embryos and the resulting reporter expression patterns are analyzed at desired timepoints during development. This system is amenable to large-scale application, facilitating rapid functional analysis of noncoding sequences from both mammalian and teleost species.