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Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases.
Remy, Séverine; Tesson, Laurent; Menoret, Séverine; Usal, Claire; De Cian, Anne; Thepenier, Virginie; Thinard, Reynald; Baron, Daniel; Charpentier, Marine; Renaud, Jean-Baptiste; Buelow, Roland; Cost, Gregory J; Giovannangeli, Carine; Fraichard, Alexandre; Concordet, Jean-Paul; Anegon, Ignacio.
Afiliación
  • Remy S; INSERM UMR 1064-ITUN, CHU de Nantes, Nantes F44093, France; Platform Rat Transgenesis, Nantes F44093, France;
  • Tesson L; INSERM UMR 1064-ITUN, CHU de Nantes, Nantes F44093, France; Platform Rat Transgenesis, Nantes F44093, France;
  • Menoret S; INSERM UMR 1064-ITUN, CHU de Nantes, Nantes F44093, France; Platform Rat Transgenesis, Nantes F44093, France;
  • Usal C; INSERM UMR 1064-ITUN, CHU de Nantes, Nantes F44093, France; Platform Rat Transgenesis, Nantes F44093, France;
  • De Cian A; INSERM U565, CNRS UMR7196, Museum National d'Histoire Naturelle, F75005 Paris, France;
  • Thepenier V; INSERM UMR 1064-ITUN, CHU de Nantes, Nantes F44093, France; Platform Rat Transgenesis, Nantes F44093, France;
  • Thinard R; INSERM UMR 1064-ITUN, CHU de Nantes, Nantes F44093, France; Platform Rat Transgenesis, Nantes F44093, France;
  • Baron D; INSERM UMR 1064-ITUN, CHU de Nantes, Nantes F44093, France;
  • Charpentier M; INSERM U565, CNRS UMR7196, Museum National d'Histoire Naturelle, F75005 Paris, France;
  • Renaud JB; INSERM U565, CNRS UMR7196, Museum National d'Histoire Naturelle, F75005 Paris, France;
  • Buelow R; Open Monoclonal Technologies, Palo Alto, California 94303, USA;
  • Cost GJ; Sangamo BioSciences, Richmond, California 94804, USA;
  • Giovannangeli C; INSERM U565, CNRS UMR7196, Museum National d'Histoire Naturelle, F75005 Paris, France;
  • Fraichard A; genOway, Lyon F69007, France.
  • Concordet JP; INSERM U565, CNRS UMR7196, Museum National d'Histoire Naturelle, F75005 Paris, France;
  • Anegon I; INSERM UMR 1064-ITUN, CHU de Nantes, Nantes F44093, France; Platform Rat Transgenesis, Nantes F44093, France;
Genome Res ; 24(8): 1371-83, 2014 Aug.
Article en En | MEDLINE | ID: mdl-24989021
The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Ingeniería Genética / Marcación de Gen Límite: Animals Idioma: En Revista: Genome Res Asunto de la revista: BIOLOGIA MOLECULAR / GENETICA Año: 2014 Tipo del documento: Article Pais de publicación: Estados Unidos
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Ingeniería Genética / Marcación de Gen Límite: Animals Idioma: En Revista: Genome Res Asunto de la revista: BIOLOGIA MOLECULAR / GENETICA Año: 2014 Tipo del documento: Article Pais de publicación: Estados Unidos