1. Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases.
- Author
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Remy S, Tesson L, Menoret S, Usal C, De Cian A, Thepenier V, Thinard R, Baron D, Charpentier M, Renaud JB, Buelow R, Cost GJ, Giovannangeli C, Fraichard A, Concordet JP, and Anegon I
- Subjects
- Animals, Base Sequence, Cells, Cultured, DNA Restriction Enzymes biosynthesis, DNA Restriction Enzymes genetics, Female, Hypoxanthine Phosphoribosyltransferase genetics, Male, Microinjections, Rats, Sprague-Dawley, Rats, Transgenic, Recombinant Proteins biosynthesis, Recombinant Proteins genetics, Recombinational DNA Repair, Zygote, Gene Targeting, Genetic Engineering
- 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., (© 2014 Remy et al.; Published by Cold Spring Harbor Laboratory Press.)
- Published
- 2014
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