西亚试剂：Knockout Rats via Embryo Microinjection of Zinc-Finger Nucl

Knockout Rats via Embryo Microinjection of Zinc-Finger Nucleases

Aron M. Geurts,^1,2,* Gregory J. Cost,^3,* Yevgeniy Freyvert,³ Bryan Zeitler,³ Jeffrey C. Miller,³ Vivian M. Choi,³ Shirin S. Jenkins,³ Adam Wood,⁴ Xiaoxia Cui,⁴ Xiangdong Meng,³ Anna Vincent,³ Stephen Lam,³ Mieczyslaw Michalkiewicz,^1,2 Rebecca Schilling,^1,2 Jamie Foeckler,³ Shawn Kalloway,³ Hartmut Weiler,^1,2 Séverine Ménoret,⁵ Ignacio Anegon,⁵ Gregory D. Davis,⁴ Lei Zhang,³ Edward J. Rebar,³ Philip D. Gregory,³ Fyodor D. Urnov,³ Howard J. Jacob,^1,2,6,$Roland Buelow^5,$

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.

1 Human and Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, WI  52336, USA.
2 Department of Physiology, Medical College of Wisconsin, Milwaukee, WI 52336, USA.
3 Sangamo BioSciences, Incorporated, Richmond, CA 94804, USA.
4 Sigma-Aldrich Biotechnology, St. Louis, MO 63103, USA.
5 INSERM, UMR 643, CHU, Nantes, Université de Nantes, 44322 Nantes, France.
6 Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI 52336, USA.
7 Open Monoclonal Technology, Incorporated, Palo Alto, CA 94303, USA.
$ To whom correspondence should be addressed
* These authors contributed equally to this work.

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