title: Site-directed zebrafish transgenesis into single landing sites with the phiC31 integrase system.
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.
authors.affiliation: Howard Hughes Medical Institute, Boston, MA 02115, USA.|Stem Cell Program, Children's Hospital Boston, Boston, MA 02115, USA.|Division of Hematology/Oncology, Children's Hospital Boston, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Christian
authors.initials: C
authors.lastname: Mosimann
authors.affiliation: Howard Hughes Medical Institute, Boston, MA 02115, USA.|Stem Cell Program, Children's Hospital Boston, Boston, MA 02115, USA.|Division of Hematology/Oncology, Children's Hospital Boston, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Ann-Christin
authors.initials: AC
authors.lastname: Puller
authors.affiliation: Howard Hughes Medical Institute, Boston, MA 02115, USA.|Stem Cell Program, Children's Hospital Boston, Boston, MA 02115, USA.|Division of Hematology/Oncology, Children's Hospital Boston, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Katy L
authors.initials: KL
authors.lastname: Lawson
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Patrick
authors.initials: P
authors.lastname: Tschopp
authors.affiliation: Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02319, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Amsterdam
authors.affiliation: Howard Hughes Medical Institute, Boston, MA 02115, USA.|Stem Cell Program, Children's Hospital Boston, Boston, MA 02115, USA.|Division of Hematology/Oncology, Children's Hospital Boston, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Leonard I
authors.initials: LI
authors.lastname: Zon
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D018014:Gene Transfer Techniques; D019426:Integrases / Q000235:genetics*; D015027:Zebrafish / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics*
chemical_list: D029961:Zebrafish Proteins; D019426:Integrases
keywords: genetics; phiC31; transgenesis; zebrafish