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Evaluation of OPEN Zinc finger nucleases for direct gene targeting of the ROSA26 locus in mouse embryos


Hermann, Mario; Maeder, Morgan L; Rector, Kyle; Ruiz, Joseph; Becher, Burkhard; Bürki, Kurt; Khayter, Cyd; Aguzzi, Adriano; Joung, J Keith; Buch, Thorsten; Pelczar, Pawel (2012). Evaluation of OPEN Zinc finger nucleases for direct gene targeting of the ROSA26 locus in mouse embryos. PLoS ONE, 7(9):e41796.

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.

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.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Institute of Neuropathology
05 Vetsuisse Faculty > Institute of Laboratory Animal Science
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:September 2012
Deposited On:19 Sep 2012 08:52
Last Modified:28 Aug 2017 10:36
Publisher:Public Library of Science (PLoS)
ISSN:1932-6203
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.1371/journal.pone.0041796
PubMed ID:22970113

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Licence: Creative Commons: Attribution 4.0 International (CC BY 4.0)