title: Homologous recombination contributes to the repair of zinc-finger-nuclease induced double strand breaks in pig primary cells and facilitates recombination with exogenous DNA.
abstract: Site-specific nucleases have become powerful tools for genome editing by the introduction of end-joining-mediated mutations, but it is unclear to which extent induced double strand breaks will also facilitate homologous recombination with exogenous DNA. This question is, however, of particular importance for somatic cells, which have to be modified for the generation of large animal models, but, on the other hand, have also been described to be reluctant to recombination-based DNA repair. Here, we examined zinc-finger nucleases for their potential to introduce modifications in pig somatic cells via end-joining or recombination. We found that co-transfection with nuclease-encoding plasmids resulted in a dramatic boost of recombination with different targeting vectors, suggesting a much more prominent role of this repair pathway in somatic cells than was previously thought. Although recombination with any of the vectors even occurred on both alleles of the target gene, we found also evidence for distinct properties of the used vectors regarding their preference for mono-allelic or bi-allelic modification. Thus, we show that the combined usage of site-specific nucleases and targeting vectors does not only promote homologous recombination in somatic cells but might also resemble a promising tool for detailed examination of DNA repair pathways.
authors.affiliation: Chair for Molecular Animal Breeding and Biotechnology, Department of Veterinary Sciences of the Ludwig-Maximilian University, Hackerstrasse 27, 85764 Oberschleissheim, Germany. Electronic address: n.klymiuk@gen.vetmed.uni-muenchen.de.
authors.forename: Nikolai
authors.initials: N
authors.lastname: Klymiuk
authors.affiliation: Chair for Molecular Animal Breeding and Biotechnology, Department of Veterinary Sciences of the Ludwig-Maximilian University, Hackerstrasse 27, 85764 Oberschleissheim, Germany.
authors.forename: Pauline
authors.initials: P
authors.lastname: Fezert
authors.affiliation: Chair for Molecular Animal Breeding and Biotechnology, Department of Veterinary Sciences of the Ludwig-Maximilian University, Hackerstrasse 27, 85764 Oberschleissheim, Germany.
authors.forename: Annegret
authors.initials: A
authors.lastname: Wünsch
authors.affiliation: Chair for Molecular Animal Breeding and Biotechnology, Department of Veterinary Sciences of the Ludwig-Maximilian University, Hackerstrasse 27, 85764 Oberschleissheim, Germany.
authors.forename: Mayuko
authors.initials: M
authors.lastname: Kurome
authors.affiliation: Chair for Molecular Animal Breeding and Biotechnology, Department of Veterinary Sciences of the Ludwig-Maximilian University, Hackerstrasse 27, 85764 Oberschleissheim, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Kessler
authors.affiliation: Chair for Molecular Animal Breeding and Biotechnology, Department of Veterinary Sciences of the Ludwig-Maximilian University, Hackerstrasse 27, 85764 Oberschleissheim, Germany; Laboratory for Functional Genome Analysis, Gene Center of the Ludwig-Maximilian University, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.
authors.forename: Eckhard
authors.initials: E
authors.lastname: Wolf
mesh_terms: D000818:Animals; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D003851:Deoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000166:cytology; D008297:Male; D053652:Nuclear Transfer Techniques; D059767:Recombinational DNA Repair; D013552:Swine; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D004247:DNA; D003851:Deoxyribonucleases
keywords: Double strand break repair; Homologous recombination; Large animal models; Site-specific nucleases; Somatic cells