Knockout rats via embryo microinjection of zinc-finger nucleases.
Journal: 2009/September - Science
ISSN: 1095-9203
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.
Relations:
Content
Citations
(348)
References
(5)
Chemicals
(4)
Organisms
(2)
Processes
(2)
Anatomy
(1)
Affiliates
(1)
Similar articles
Articles by the same authors
Discussion board
Science 325(5939): 433

Knockout Rats Produced Using Designed Zinc Finger Nucleases

+17 authors

Supplementary Material

supplement methods, figs and table

supplement methods, figs and table

Click here to view.(1.1M, pdf)

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, Inc. 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.

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
Sangamo BioSciences, Inc., Richmond, CA 94804, USA
Sigma-Aldrich Biotecnhnology, St. Louis, MO 63103, USA
INSERM, UMR 643; CHU, Nantes; Université de Nantes, Nantes, France
Open Monoclonal Technology, Inc., Palo Alto, CA 94303, USA
Corresponding author.
Howard J. Jacob: ude.wcm@bocaj; Roland Buelow: ten.cnitmo@woleubr
Both authors contributed equally.
Howard J. Jacob: ude.wcm@bocaj; Roland Buelow: ten.cnitmo@woleubr

Abstract

The toolbox of rat genetics currently lacks the ability to introduce site-directed, heritable mutations into the genome to create knockout animals. 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 mRNA encoding ZFNs into the one-cell rat embryo leads to a high frequency of animals carrying 25–100% 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 four months time, paving the way to a humanized monoclonal antibody platform and additional human disease models.

Abstract

The laboratory rat is a well-established model for the genetic dissection of human disease-related traits (1) despite the fact that targeted modification of its genome is largely intractable. We investigated the application of engineered zinc-finger nucleases (ZFNs;(2)) for the elimination of specific rat gene function and generation of “knockout” rats. ZFNs induce site-specific, double-strand DNA breaks that can be repaired by the error-prone non-homologous end joining DNA repair pathway to result in a targeted mutation (Fig. 1A). In the fruit fly and zebrafish, direct embryo injection of ZFN-encoding mRNA has been used to generate heritable knockout mutations at specific loci (2).

An external file that holds a picture, illustration, etc.
Object name is nihms171136f1.jpg

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.

The design and validation of ZFN reagents to target a single-copy Green Fluorescent Protein (GFP) transgene inserted in a rat chromosome and two endogenous rat genes, IgM and Rab38, was performed as described (3) and is detailed in the supplemental materials. To take advantage of the potential for greater specificity-of-action afforded by longer (and therefore rarer) targets, we used 5- and 6- finger ZFNs.

We delivered these ZFNs to 36 hemizygous GFP-transgenic (4) inbred SS (Dahl S), 91 inbred FHH (Fawn-hooded hypertensive), and 2793 outbred SD (Sprague Dawley) embryos by pronuclear or intracytoplasmic injection of ZFN-encoding DNA or mRNA at different concentrations (Fig. 1D). Screening 295 founder animals yielded 35 (12%) that harbored targeted mutations.

Full knockout of the GFP transgene was achieved, as mutant animals lacked both GFP expression and wild-type GFP sequence (Fig. 1, B and C). Thirty-two IgM mutants and the single Rab38 mutant carried 25–100% disrupted target chromosomes (Fig. S1). Sequence analysis of 18 founders revealed deletion alleles ranging from 3–187 base pairs; of note, one animal carried biallelic mutations in IgM (Table S1). Furthermore, ZFN-mediated gene disruption demonstrated high fidelity for each target sequence since no ZFN-induced mutations were detected in target-gene-disrupted animals at any of 20 predicted ZFN off-target sites (Figs. S2, S3). After breeding to wild-type animals, 1 out of 1 GFP and 3 out of 4 IgM mutations were transmitted through the germline, one of which was subsequently bred to homozygosity (Figs. 1D, S4).

The high percentage of disrupted chromosomes demonstrates that ZFNs are active in early rat embryos from three strains, leading to both mono- and biallelic gene disruption. Although we observed no cleavage at predicted off-target sites, such events could be segregated away from the desired mutation by backcrossing to the parental strain. ZFN-driven gene disruption and germline transmission can be accomplished in 4 months’ time, and ZFNs can be engineered against a broad range of sequences (5, 6). Hence this strategy adds a valuable tool to an increasingly powerful rat genetic tool box, opening up a range of new experiments and models of human disease.

References

  • 1. Aitman TJ, et al Nat Genet. 2008 May;40:516.[PubMed][Google Scholar]
  • 2. Carroll D. Gene Ther. 2008 Nov;15:1463.
  • 3. Doyon Y, et al Nat Biotech. 2008 Jun;26:702.[Google Scholar]
  • 4. Michalkiewicz M, et al Am J Physiol Heart Circ Physiol. 2007 Jul;293:H881.[PubMed][Google Scholar]
  • 5. Pabo CO, Peisach E, Grant RA. Annu Rev Biochem. 2001;70:313.[PubMed]
  • 6. Klug A. Proc Jpn Acad. 2005;81:87.[PubMed]
Collaboration tool especially designed for Life Science professionals.Drag-and-drop any entity to your messages.