Background: Linear DNA-based and Tol2-mediated transgenesis are powerful tools for the generation of transgenic zebrafish. However, the integration of multiple copies or transgenes at random genomic locations complicates comparative transgene analysis and makes long-term transgene stability unpredictable with variable expression. Targeted, site-directed transgene integration into pre-determined genomic loci can circumvent these issues. The phiC31 integrase catalyzes the unidirectional recombination reaction between heterotypic attP and attB sites and is an efficient platform for site-directed transgenesis.
Results: We report the implementation of the phiC31 integrase-mediated attP/attB recombination for site-directed zebrafish transgenics of attB-containing transgene vectors into single genomic attP landing sites. We generated Tol2-based single-insertion attP transgenic lines and established their performance in phiC31 integrase-catalyzed integration of an attB-containing transgene vector. We found stable germline transmission into the next generation of an attB reporter transgene in 34% of all tested animals. We further characterized two functional attP landing site lines and determined their genomic location. Our experiments also demonstrate tissue-specific transgene applications as well as long-term stability of phiC31-mediated transgenes.
Conclusions: Our results establish phiC31 integrase-controlled site-directed transgenesis into single, genomic attP sites as space-, time-, and labor-efficient zebrafish transgenesis technique. The described reagents are available for distribution to the zebrafish community.
Keywords: genetics; phiC31; transgenesis; zebrafish.
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