Christian Mosimann1,2,3, Ann-Christin Puller1,2,3, Katy L Lawson1,2,3, Patrick Tschopp4, Adam Amsterdam5, Leonard I Zon1,2,3. 1. Howard Hughes Medical Institute, Boston, MA 02115, USA. 2. Stem Cell Program, Children's Hospital Boston, Boston, MA 02115, USA. 3. Division of Hematology/Oncology, Children's Hospital Boston, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115, USA. 4. Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. 5. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02319, USA.
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.
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.
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