Targeted transgene integration in plant cells using designed zinc finger nucleases

Plant Mol Biol. 2009 Apr;69(6):699-709. doi: 10.1007/s11103-008-9449-7. Epub 2008 Dec 27.

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.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Binding Sites
  • Cells, Cultured
  • Chitinases / genetics
  • Endonucleases / genetics
  • Endonucleases / metabolism*
  • Glucuronidase / genetics
  • Glucuronidase / metabolism
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Molecular Sequence Data
  • Nicotiana / cytology
  • Nicotiana / genetics
  • Nicotiana / metabolism
  • Polymerase Chain Reaction
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Recombination, Genetic
  • Transfection / methods
  • Transgenes / genetics*
  • Zinc Fingers / genetics*

Substances

  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • Endonucleases
  • Chitinases
  • Glucuronidase