title: Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases.
abstract: 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.
authors.affiliation: INSERM UMR 1064-ITUN, CHU de Nantes, Nantes F44093, France; Platform Rat Transgenesis, Nantes F44093, France;
authors.forename: Séverine
authors.initials: S
authors.lastname: Remy
authors.affiliation: INSERM UMR 1064-ITUN, CHU de Nantes, Nantes F44093, France; Platform Rat Transgenesis, Nantes F44093, France;
authors.forename: Laurent
authors.initials: L
authors.lastname: Tesson
authors.affiliation: INSERM UMR 1064-ITUN, CHU de Nantes, Nantes F44093, France; Platform Rat Transgenesis, Nantes F44093, France;
authors.forename: Séverine
authors.initials: S
authors.lastname: Menoret
authors.affiliation: INSERM UMR 1064-ITUN, CHU de Nantes, Nantes F44093, France; Platform Rat Transgenesis, Nantes F44093, France;
authors.forename: Claire
authors.initials: C
authors.lastname: Usal
authors.affiliation: INSERM U565, CNRS UMR7196, Museum National d'Histoire Naturelle, F75005 Paris, France;
authors.forename: Anne
authors.initials: A
authors.lastname: De Cian
authors.affiliation: INSERM UMR 1064-ITUN, CHU de Nantes, Nantes F44093, France; Platform Rat Transgenesis, Nantes F44093, France;
authors.forename: Virginie
authors.initials: V
authors.lastname: Thepenier
authors.affiliation: INSERM UMR 1064-ITUN, CHU de Nantes, Nantes F44093, France; Platform Rat Transgenesis, Nantes F44093, France;
authors.forename: Reynald
authors.initials: R
authors.lastname: Thinard
authors.affiliation: INSERM UMR 1064-ITUN, CHU de Nantes, Nantes F44093, France;
authors.forename: Daniel
authors.initials: D
authors.lastname: Baron
authors.affiliation: INSERM U565, CNRS UMR7196, Museum National d'Histoire Naturelle, F75005 Paris, France;
authors.forename: Marine
authors.initials: M
authors.lastname: Charpentier
authors.affiliation: INSERM U565, CNRS UMR7196, Museum National d'Histoire Naturelle, F75005 Paris, France;
authors.forename: Jean-Baptiste
authors.initials: JB
authors.lastname: Renaud
authors.affiliation: Open Monoclonal Technologies, Palo Alto, California 94303, USA;
authors.forename: Roland
authors.initials: R
authors.lastname: Buelow
authors.affiliation: Sangamo BioSciences, Richmond, California 94804, USA;
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Cost
authors.affiliation: INSERM U565, CNRS UMR7196, Museum National d'Histoire Naturelle, F75005 Paris, France;
authors.forename: Carine
authors.initials: C
authors.lastname: Giovannangeli
authors.affiliation: genOway, Lyon F69007, France.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Fraichard
authors.affiliation: INSERM U565, CNRS UMR7196, Museum National d'Histoire Naturelle, F75005 Paris, France;
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Concordet
authors.affiliation: INSERM UMR 1064-ITUN, CHU de Nantes, Nantes F44093, France; Platform Rat Transgenesis, Nantes F44093, France;
authors.forename: Ignacio
authors.initials: I
authors.lastname: Anegon
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004262:DNA Restriction Enzymes / Q000096:biosynthesis / Q000235:genetics; D005260:Female; D018390:Gene Targeting; D005818:Genetic Engineering; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008297:Male; D008845:Microinjections; D017207:Rats, Sprague-Dawley; D055647:Rats, Transgenic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D059767:Recombinational DNA Repair; D015053:Zygote
chemical_list: D011994:Recombinant Proteins; D007041:Hypoxanthine Phosphoribosyltransferase; D004262:DNA Restriction Enzymes