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Plant Molecular Biology

, Volume 69, Issue 6, pp 699–709 | Cite as

Targeted transgene integration in plant cells using designed zinc finger nucleases

  • Charles Q. Cai
  • Yannick Doyon
  • W. Michael Ainley
  • Jeffrey C. Miller
  • Russell C. DeKelver
  • Erica A. Moehle
  • Jeremy M. Rock
  • Ya-Li Lee
  • Robbi Garrison
  • Lisa Schulenberg
  • Ryan Blue
  • Andrew Worden
  • Lisa Baker
  • Farhoud Faraji
  • Lei Zhang
  • Michael C. Holmes
  • Edward J. Rebar
  • Trevor N. Collingwood
  • Beth Rubin-Wilson
  • Philip D. Gregory
  • Fyodor D. Urnov
  • Joseph F. Petolino
Article

Abstract

Targeted transgene integration in plants remains a significant technical challenge for both basic and applied research. Here it is reported that designed zinc finger nucleases (ZFNs) can drive site-directed DNA integration into transgenic and native gene loci. A dimer of designed 4-finger ZFNs enabled intra-chromosomal reconstitution of a disabled gfp reporter gene and site-specific transgene integration into chromosomal reporter loci following co-transformation of tobacco cell cultures with a donor construct comprised of sequences necessary to complement a non-functional pat herbicide resistance gene. In addition, a yeast-based assay was used to identify ZFNs capable of cleaving a native endochitinase gene. Agrobacterium delivery of a Ti plasmid harboring both the ZFNs and a donor DNA construct comprising a pat herbicide resistance gene cassette flanked by short stretches of homology to the endochitinase locus yielded up to 10% targeted, homology-directed transgene integration precisely into the ZFN cleavage site. Given that ZFNs can be designed to recognize a wide range of target sequences, these data point toward a novel approach for targeted gene addition, replacement and trait stacking in plants.

Keywords

Double strand DNA breaks Homology-directed repair Site-directed transgene integration Zinc finger nucleases 

Notes

Acknowledgments

Thanks to Sangamo’s production group for zinc finger protein assembly and Aaron Klug and Vipula Shukla for helpful comments on the manuscript.

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Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Charles Q. Cai
    • 1
  • Yannick Doyon
    • 2
  • W. Michael Ainley
    • 1
  • Jeffrey C. Miller
    • 2
  • Russell C. DeKelver
    • 2
  • Erica A. Moehle
    • 2
  • Jeremy M. Rock
    • 2
  • Ya-Li Lee
    • 2
  • Robbi Garrison
    • 1
  • Lisa Schulenberg
    • 1
  • Ryan Blue
    • 1
  • Andrew Worden
    • 1
  • Lisa Baker
    • 1
  • Farhoud Faraji
    • 2
  • Lei Zhang
    • 2
  • Michael C. Holmes
    • 2
  • Edward J. Rebar
    • 2
  • Trevor N. Collingwood
    • 2
  • Beth Rubin-Wilson
    • 1
  • Philip D. Gregory
    • 2
  • Fyodor D. Urnov
    • 2
  • Joseph F. Petolino
    • 1
  1. 1.Dow AgroSciences, LLCIndianapolisUSA
  2. 2.Sangamo BioSciencesRichmondUSA

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