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Knockout Rats via Embryo Microinjection of Zinc-Finger Nucleases

Aron M. Geurts, 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 [email protected], and Roland Buelow [email protected]Authors Info & Affiliations
Science
24 Jul 2009
Vol 325, Issue 5939
p. 433
DOI: 10.1126/science.1172447
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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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Supplementary Material

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Information & Authors

Information

Published In

Science
Volume 325 | Issue 5939
24 July 2009

Copyright

Copyright © 2009, American Association for the Advancement of Science.

Submission history

Received: 18 February 2009
Accepted: 1 May 2009
Published in print: 24 July 2009

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Acknowledgments

We thank R. Jaenisch, R. Hammer, P. Sullivan, and three anonymous referees for helpful suggestions; D. Smoller and E. Lanphier for support; E. Eastlund for the Rab38 ZFN mRNA; R. DeKelver and R. Amora for technical assistance; and Caliper Life Sciences, Incorporated for excellent service. Supported by NIH grants 5U01HL066579-08 and 5P01HL082798-03, a sponsored research agreement between the Medical College of Wisconsin and Sigma-Aldrich, and the American Physiological Society Fellowship in Physiological Genomics to A.M.G. The authors are filing patents based on the results reported in this paper.

Authors

Affiliations

Aron M. Geurts*
Human and Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, WI 52336, USA.
Department of Physiology, Medical College of Wisconsin, Milwaukee, WI 52336, USA.
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Gregory J. Cost*
Sangamo BioSciences, Incorporated, Richmond, CA 94804, USA.
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Yevgeniy Freyvert
Sangamo BioSciences, Incorporated, Richmond, CA 94804, USA.
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Bryan Zeitler
Sangamo BioSciences, Incorporated, Richmond, CA 94804, USA.
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Jeffrey C. Miller
Sangamo BioSciences, Incorporated, Richmond, CA 94804, USA.
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Vivian M. Choi
Sangamo BioSciences, Incorporated, Richmond, CA 94804, USA.
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Shirin S. Jenkins
Sangamo BioSciences, Incorporated, Richmond, CA 94804, USA.
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Adam Wood
Sigma-Aldrich Biotechnology, St. Louis, MO 63103, USA.
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Xiaoxia Cui
Sigma-Aldrich Biotechnology, St. Louis, MO 63103, USA.
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Xiangdong Meng
Sangamo BioSciences, Incorporated, Richmond, CA 94804, USA.
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Anna Vincent
Sangamo BioSciences, Incorporated, Richmond, CA 94804, USA.
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Stephen Lam
Sangamo BioSciences, Incorporated, Richmond, CA 94804, USA.
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Mieczyslaw Michalkiewicz
Human and Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, WI 52336, USA.
Department of Physiology, Medical College of Wisconsin, Milwaukee, WI 52336, USA.
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Rebecca Schilling
Human and Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, WI 52336, USA.
Department of Physiology, Medical College of Wisconsin, Milwaukee, WI 52336, USA.
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Jamie Foeckler
Sangamo BioSciences, Incorporated, Richmond, CA 94804, USA.
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Shawn Kalloway
Sangamo BioSciences, Incorporated, Richmond, CA 94804, USA.
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Hartmut Weiler
Human and Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, WI 52336, USA.
Department of Physiology, Medical College of Wisconsin, Milwaukee, WI 52336, USA.
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Séverine Ménoret
INSERM, UMR 643, CHU, Nantes, Université de Nantes, 44322 Nantes, France.
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Ignacio Anegon
INSERM, UMR 643, CHU, Nantes, Université de Nantes, 44322 Nantes, France.
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Gregory D. Davis
Sigma-Aldrich Biotechnology, St. Louis, MO 63103, USA.
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Lei Zhang
Sangamo BioSciences, Incorporated, Richmond, CA 94804, USA.
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Edward J. Rebar
Sangamo BioSciences, Incorporated, Richmond, CA 94804, USA.
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Philip D. Gregory
Sangamo BioSciences, Incorporated, Richmond, CA 94804, USA.
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Fyodor D. Urnov
Sangamo BioSciences, Incorporated, Richmond, CA 94804, USA.
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Howard J. Jacob [email protected]
Human and Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, WI 52336, USA.
Department of Physiology, Medical College of Wisconsin, Milwaukee, WI 52336, USA.
Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI 52336, USA.
To whom correspondence should be addressed. E-mail: [email protected] (H.J.J.); [email protected] (R.B.)
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Roland Buelow [email protected]
Open Monoclonal Technology, Incorporated, Palo Alto, CA 94303, USA.
To whom correspondence should be addressed. E-mail: [email protected] (H.J.J.); [email protected] (R.B.)
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Notes

*
These authors contributed equally to this work.

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  • Aron M. Geurts et al.
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Knockout Rats via Embryo Microinjection of Zinc-Finger Nucleases.Science325,433-433(2009).DOI:10.1126/science.1172447

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