title: Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases.
abstract: The frog Xenopus, an important research organism in cell and developmental biology, currently lacks tools for targeted mutagenesis. Here, we address this problem by genome editing with zinc-finger nucleases (ZFNs). ZFNs directed against an eGFP transgene in Xenopus tropicalis induced mutations consistent with nonhomologous end joining at the target site, resulting in mosaic loss of the fluorescence phenotype at high frequencies. ZFNs directed against the noggin gene produced tadpoles and adult animals carrying up to 47% disrupted alleles, and founder animals yielded progeny carrying insertions and deletions in the noggin gene with no indication of off-target effects. Furthermore, functional tests demonstrated an allelic series of activity between three germ-line mutant alleles. Because ZFNs can be designed against any locus, our data provide a generally applicable protocol for gene disruption in Xenopus.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA.
authors.forename: John J
authors.initials: JJ
authors.lastname: Young
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Cherone
authors.forename: Yannick
authors.initials: Y
authors.lastname: Doyon
authors.forename: Irina
authors.initials: I
authors.lastname: Ankoudinova
authors.forename: Farhoud M
authors.initials: FM
authors.lastname: Faraji
authors.forename: Andrew H
authors.initials: AH
authors.lastname: Lee
authors.forename: Catherine
authors.initials: C
authors.lastname: Ngo
authors.forename: Dmitry Y
authors.initials: DY
authors.lastname: Guschin
authors.forename: David E
authors.initials: DE
authors.lastname: Paschon
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Miller
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Rebar
authors.forename: Philip D
authors.initials: PD
authors.lastname: Gregory
authors.forename: Fyodor D
authors.initials: FD
authors.lastname: Urnov
authors.forename: Richard M
authors.initials: RM
authors.lastname: Harland
authors.forename: Bryan
authors.initials: B
authors.lastname: Zeitler
mesh_terms: D000483:Alleles; D000818:Animals; D030801:Animals, Genetically Modified; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003851:Deoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods*; D014981:Xenopus; D029867:Xenopus Proteins / Q000235:genetics* / Q000378:metabolism; D016335:Zinc Fingers; D000099176:Noggin Protein
chemical_list: D002352:Carrier Proteins; D003851:Deoxyribonucleases; D029867:Xenopus Proteins; D000099176:Noggin Protein