title: Efficient genome engineering by targeted homologous recombination in mouse embryos using transcription activator-like effector nucleases.
abstract: Generation of mouse models by introducing transgenes using homologous recombination is critical for understanding fundamental biology and pathology of human diseases. Here we investigate whether artificial transcription activator-like effector nucleases (TALENs)-powerful tools that induce DNA double-strand breaks at specific genomic locations-can be combined with a targeting vector to induce homologous recombination for the introduction of a transgene in embryonic stem cells and fertilized murine oocytes. We describe the generation of a conditional mouse model using TALENs, which introduce double-strand breaks at the genomic locus of the special AT-rich sequence-binding protein-1 in combination with a large 14.4 kb targeting template vector. We report successful germline transmission of this allele and demonstrate its recombination in primary cells in the presence of Cre-recombinase. These results suggest that TALEN-assisted induction of DNA double-strand breaks can facilitate homologous recombination of complex targeting constructs directly in oocytes.
authors.affiliation: Genomics and Immunoregulation, LIMES Institute, University of Bonn, Carl-Troll-Strasse 31, D-53115 Bonn, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Sommer
authors.affiliation: Genomics and Immunoregulation, LIMES Institute, University of Bonn, Carl-Troll-Strasse 31, D-53115 Bonn, Germany.
authors.forename: Annika
authors.initials: A
authors.lastname: Peters
authors.affiliation: 1] Genomics and Immunoregulation, LIMES Institute, University of Bonn, Carl-Troll-Strasse 31, D-53115 Bonn, Germany [2].
authors.forename: Tristan
authors.initials: T
authors.lastname: Wirtz
authors.affiliation: Genomics and Immunoregulation, LIMES Institute, University of Bonn, Carl-Troll-Strasse 31, D-53115 Bonn, Germany.
authors.forename: Maren
authors.initials: M
authors.lastname: Mai
authors.affiliation: Max Planck Institute for Neurological Research and Institute for Genetics, University of Cologne, Gleuelerstrasse 50, D-50931 Cologne, Germany.
authors.forename: Justus
authors.initials: J
authors.lastname: Ackermann
authors.affiliation: Genomics and Immunoregulation, LIMES Institute, University of Bonn, Carl-Troll-Strasse 31, D-53115 Bonn, Germany.
authors.forename: Yasser
authors.initials: Y
authors.lastname: Thabet
authors.affiliation: Department of Experimental Therapy, University Hospital Bonn, Sigmund-Freud-Strasse 25, D-53105 Bonn, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Schmidt
authors.affiliation: Immunology and Environment, LIMES Institute, University of Bonn, Carl-Troll-Strasse 31, D-53115 Bonn, Germany.
authors.forename: Heike
authors.initials: H
authors.lastname: Weighardt
authors.affiliation: Max Planck Institute for Neurological Research and Institute for Genetics, University of Cologne, Gleuelerstrasse 50, D-50931 Cologne, Germany.
authors.forename: F Thomas
authors.initials: FT
authors.lastname: Wunderlich
authors.affiliation: Genomic Resource Centre, LIMES Institute, University of Bonn, Carl-Troll-Strasse 31, D-53115 Bonn, Germany.
authors.forename: Joachim
authors.initials: J
authors.lastname: Degen
authors.affiliation: Genomics and Immunoregulation, LIMES Institute, University of Bonn, Carl-Troll-Strasse 31, D-53115 Bonn, Germany.
authors.forename: Joachim L
authors.initials: JL
authors.lastname: Schultze
authors.affiliation: Genomics and Immunoregulation, LIMES Institute, University of Bonn, Carl-Troll-Strasse 31, D-53115 Bonn, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Beyer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics; D003851:Deoxyribonucleases / Q000235:genetics* / Q000502:physiology*; D004622:Embryo, Mammalian / Q000166:cytology* / Q000502:physiology; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology; D019426:Integrases / Q000502:physiology; D036961:Matrix Attachment Region Binding Proteins / Q000235:genetics / Q000502:physiology; D051379:Mice; D023421:Models, Animal; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D009865:Oocytes / Q000166:cytology / Q000502:physiology; D011995:Recombination, Genetic / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics* / Q000502:physiology*
chemical_list: D036961:Matrix Attachment Region Binding Proteins; C469541:Satb1 protein, mouse; D004247:DNA; C045073:Cre recombinase; D019426:Integrases; D003851:Deoxyribonucleases