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Dev Dyn. 2013 Aug;242(8):949-963. doi: 10.1002/dvdy.23989. Epub 2013 Jul 3.

Site-directed zebrafish transgenesis into single landing sites with the phiC31 integrase system.

Author information

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.
#
Contributed equally

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.

KEYWORDS:

genetics; phiC31; transgenesis; zebrafish

PMID:
23723152
PMCID:
PMC3775328
DOI:
10.1002/dvdy.23989
[Indexed for MEDLINE]
Free PMC Article

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