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Link to original content: http://www.ncbi.nlm.nih.gov/pubmed/19628861
Knockout rats via embryo microinjection of zinc-finger nucleases - PubMed Skip to main page content
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. 2009 Jul 24;325(5939):433.
doi: 10.1126/science.1172447.

Knockout rats via embryo microinjection of zinc-finger nucleases

Aron M Geurts  1 , Gregory J Cost, Yevgeniy Freyvert, Bryan Zeitler, Jeffrey C Miller, Vivian M Choi, Shirin S Jenkins, Adam Wood, Xiaoxia Cui, Xiangdong Meng, Anna Vincent, Stephen Lam, Mieczyslaw Michalkiewicz, Rebecca Schilling, Jamie Foeckler, Shawn Kalloway, Hartmut Weiler, Séverine Ménoret, Ignacio Anegon, Gregory D Davis, Lei Zhang, Edward J Rebar, Philip D Gregory, Fyodor D Urnov, Howard J Jacob, Roland Buelow
Affiliations

Knockout rats via embryo microinjection of zinc-finger nucleases

Aron M Geurts et al. Science. .

Abstract

The toolbox of rat genetics currently lacks the ability to introduce site-directed, heritable mutations into the genome to create knockout animals. By using engineered zinc-finger nucleases (ZFNs) designed to target an integrated reporter and two endogenous rat genes, Immunoglobulin M (IgM) and Rab38, we demonstrate that a single injection of DNA or messenger RNA encoding ZFNs into the one-cell rat embryo leads to a high frequency of animals carrying 25 to 100% disruption at the target locus. These mutations are faithfully and efficiently transmitted through the germline. Our data demonstrate the feasibility of targeted gene disruption in multiple rat strains within 4 months time, paving the way to a humanized monoclonal antibody platform and additional human disease models.

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Figures

Fig. 1
Fig. 1
ZFN-mediated gene disruption in rat embryos. (A) ZFNs containing 5–6 fingers were designed to target coding sequences of interest (gray lines) for site-specific cleavage. (B) Two of five pups born after microinjection of GFP-targeted ZFNs were devoid of GFP expression. (C) Polymerase chain reaction using GFP-specific primers revealed truncated, but no wild-type sequence in each of the GFP negative pups compared to positive littermates. SS- Dahl S control DNA; NT indicates no template. (D) Table of injection data revealing successful mutagenesis of the three gene targets after multiple delivery methods and doses in three rat strains.
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