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Targeted integration in rat and mouse embryos with zinc-finger nucleases

Abstract

Gene targeting is indispensible for reverse genetics and the generation of animal models of disease. The mouse has become the most commonly used animal model system owing to the success of embryonic stem cell–based targeting technology1, whereas other mammalian species lack convenient tools for genome modification. Recently, microinjection of engineered zinc-finger nucleases (ZFNs) in embryos was used to generate gene knockouts in the rat2,3 and the mouse4 by introducing nonhomologous end joining (NHEJ)-mediated deletions or insertions at the target site. Here we use ZFN technology in embryos to introduce sequence-specific modifications (knock-ins) by means of homologous recombination in Sprague Dawley and Long-Evans hooded rats and FVB mice. This approach enables precise genome engineering to generate modifications such as point mutations, accurate insertions and deletions, and conditional knockouts and knock-ins. The same strategy can potentially be applied to many other species for which genetic engineering tools are needed.

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Figure 1: Targeted integration of NotI restriction site.
Figure 2: Targeted integration of a GFP cassette.
Figure 3: Germline transmission of site-specific GFP integration.

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Acknowledgements

We thank J. Books for technical assistance; R. DeKelver at Sangamo Biosciences for GFP plasmid; A. Harrington and L. Liaw for mouse microinjections; Z. Cui, G. Davis and T. Saunders for discussion; D. Carroll, K. Cunningham, T. Collingwood and F. Urnov for suggestions; and S. Fine for excellent assistance with figures.

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Authors and Affiliations

Authors

Contributions

X.C. designed the project, built donor constructs and carried out some of the diagnostic PCRs; D.J. built donor constructs and performed most of the PCR diagnosis; D.A.F. performed Southern blot analysis; Y.W. microinjected the rat samples; D.M.B. assembled ZFNs; X.C., D.J., D.A.F. and E.J.W. wrote the manuscript.

Corresponding author

Correspondence to Xiaoxia Cui.

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Competing interests

All authors are full-time employees of Sigma-Aldrich Biotechnology.

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Supplementary Tables 1–4, Supplementary Methods and Supplementary Figs. 1–9 (PDF 4052 kb)

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Cui, X., Ji, D., Fisher, D. et al. Targeted integration in rat and mouse embryos with zinc-finger nucleases. Nat Biotechnol 29, 64–67 (2011). https://doi.org/10.1038/nbt.1731

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