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- Title
Site-directed zebrafish transgenesis into single landing sites with the phiC31 integrase system.
- Authors
Mosimann, Christian; Puller, Ann‐Christin; Lawson, Katy L.; Tschopp, Patrick; Amsterdam, Adam; Zon, Leonard I.
- Abstract
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. Developmental Dynamics 242:949-963, 2013. © 2013 Wiley Periodicals, Inc.
- Publication
Developmental Dynamics, 2013, Vol 242, Issue 8, p949
- ISSN
1058-8388
- Publication type
Article
- DOI
10.1002/dvdy.23989