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Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases

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 cr...

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Autores principales: 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
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4120090/
https://www.ncbi.nlm.nih.gov/pubmed/24989021
http://dx.doi.org/10.1101/gr.171538.113
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author 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
author_facet 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
author_sort Remy, Séverine
collection PubMed
description 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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spelling pubmed-41200902015-02-01 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 Genome Res Method 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. Cold Spring Harbor Laboratory Press 2014-08 /pmc/articles/PMC4120090/ /pubmed/24989021 http://dx.doi.org/10.1101/gr.171538.113 Text en © 2014 Remy et al.; Published by Cold Spring Harbor Laboratory Press http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
spellingShingle Method
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
Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases
title Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases
title_full Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases
title_fullStr Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases
title_full_unstemmed Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases
title_short Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases
title_sort efficient gene targeting by homology-directed repair in rat zygotes using tale nucleases
topic Method
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4120090/
https://www.ncbi.nlm.nih.gov/pubmed/24989021
http://dx.doi.org/10.1101/gr.171538.113
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