title: Evaluation of OPEN zinc finger nucleases for direct gene targeting of the ROSA26 locus in mouse embryos.
abstract: Zinc finger nucleases (ZFNs) enable precise genome modification in a variety of organisms and cell types. Commercial ZFNs were reported to enhance gene targeting directly in mouse zygotes, whereas similar approaches using publicly available resources have not yet been described. Here we report precise targeted mutagenesis of the mouse genome using Oligomerized Pool Engineering (OPEN) ZFNs. OPEN ZFN can be constructed using publicly available resources and therefore provide an attractive alternative for academic researchers. Two ZFN pairs specific to the mouse genomic locus gt(ROSA26)Sor were generated by OPEN selections and used for gene disruption and homology-mediated gene replacement in single cell mouse embryos. One specific ZFN pair facilitated non-homologous end joining (NHEJ)-mediated gene disruption when expressed in mouse zygotes. We also observed a single homologous recombination (HR)-driven gene replacement event when this ZFN pair was co-injected with a targeting vector. Our experiments demonstrate the feasibility of achieving both gene ablation through NHEJ and gene replacement by HR by using the OPEN ZFN technology directly in mouse zygotes.
authors.affiliation: Institute of Laboratory Animal Science, University of Zurich, Zurich, Switzerland.
authors.forename: Mario
authors.initials: M
authors.lastname: Hermann
authors.forename: Morgan L
authors.initials: ML
authors.lastname: Maeder
authors.forename: Kyle
authors.initials: K
authors.lastname: Rector
authors.forename: Joseph
authors.initials: J
authors.lastname: Ruiz
authors.forename: Burkhard
authors.initials: B
authors.lastname: Becher
authors.forename: Kurt
authors.initials: K
authors.lastname: Bürki
authors.forename: Cyd
authors.initials: C
authors.lastname: Khayter
authors.forename: Adriano
authors.initials: A
authors.lastname: Aguzzi
authors.forename: J Keith
authors.initials: JK
authors.lastname: Joung
authors.forename: Thorsten
authors.initials: T
authors.lastname: Buch
authors.forename: Pawel
authors.initials: P
authors.lastname: Pelczar
mesh_terms: D000818:Animals; D059766:DNA End-Joining Repair / Q000235:genetics; D004622:Embryo, Mammalian / Q000378:metabolism*; D004720:Endonucleases / Q000235:genetics*; D005260:Female; D018390:Gene Targeting / Q000379:methods*; D056426:Genetic Loci / Q000235:genetics*; D059765:Homologous Recombination / Q000235:genetics; D008297:Male; D051379:Mice; D008845:Microinjections; D011506:Proteins / Q000235:genetics*; D022661:RNA, Untranslated; D016335:Zinc Fingers / Q000235:genetics*; D015053:Zygote / Q000378:metabolism
chemical_list: C110853:Gt(ROSA)26Sor non-coding RNA, mouse; D011506:Proteins; D022661:RNA, Untranslated; D004720:Endonucleases