title: Targeted transgene integration in plant cells using designed zinc finger nucleases.
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.
authors.affiliation: Dow AgroSciences, LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA.
authors.forename: Charles Q
authors.initials: CQ
authors.lastname: Cai
authors.forename: Yannick
authors.initials: Y
authors.lastname: Doyon
authors.forename: W Michael
authors.initials: WM
authors.lastname: Ainley
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Miller
authors.forename: Russell C
authors.initials: RC
authors.lastname: Dekelver
authors.forename: Erica A
authors.initials: EA
authors.lastname: Moehle
authors.forename: Jeremy M
authors.initials: JM
authors.lastname: Rock
authors.forename: Ya-Li
authors.initials: YL
authors.lastname: Lee
authors.forename: Robbi
authors.initials: R
authors.lastname: Garrison
authors.forename: Lisa
authors.initials: L
authors.lastname: Schulenberg
authors.forename: Ryan
authors.initials: R
authors.lastname: Blue
authors.forename: Andrew
authors.initials: A
authors.lastname: Worden
authors.forename: Lisa
authors.initials: L
authors.lastname: Baker
authors.forename: Farhoud
authors.initials: F
authors.lastname: Faraji
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Michael C
authors.initials: MC
authors.lastname: Holmes
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Rebar
authors.forename: Trevor N
authors.initials: TN
authors.lastname: Collingwood
authors.forename: Beth
authors.initials: B
authors.lastname: Rubin-Wilson
authors.forename: Philip D
authors.initials: PD
authors.lastname: Gregory
authors.forename: Fyodor D
authors.initials: FD
authors.lastname: Urnov
authors.forename: Joseph F
authors.initials: JF
authors.lastname: Petolino
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D002688:Chitinases / Q000235:genetics; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D014026:Nicotiana / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods; D019076:Transgenes / Q000235:genetics*; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004720:Endonucleases; D002688:Chitinases; D005966:Glucuronidase