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Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases

by Ankoudinova, Irina , Gregory, Philip D , Miller, Jeffrey C , Doyon, Yannick , Cherone, Jennifer M , Lee, Andrew H , Zhang, Lei , Ngo, Catherine , Paschon, David E , Zeitler, Bryan , Guschin, Dmitry Y , Faraji, Farhoud M , Urnov, Fyodor D , Harland, Richard M , Rebar, Edward J , Young, John J

in adults / Alleles / Amphibians / Animals / Animals, Genetically Modified / Anura / Binding sites / Biological Sciences / Carrier Proteins - genetics / Carrier Proteins - metabolism / Cells / Deoxyribonucleases - genetics / Deoxyribonucleases - metabolism / Embryos / fluorescence / Frogs / Gene expression / Gene loci / gene targeting / Gene Targeting - methods / Genetic loci / Genetic mutation / Genomes / Germ cells / loci / Messenger RNA / mutants / Mutation / nucleases / phenotype / progeny / RNA / site-directed mutagenesis / Tadpoles / Transgenes / Xenopus / Xenopus Proteins - genetics / Xenopus Proteins - metabolism / Xenopus tropicalis / Zinc / Zinc Fingers

2011

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Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases

2011

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Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases

2011

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Journal Article

Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases

Ankoudinova, Irina,

Gregory, Philip D,

Miller, Jeffrey C,

Doyon, Yannick,

Cherone, Jennifer M,

Lee, Andrew H,

Zhang, Lei,

Ngo, Catherine,

Paschon, David E,

Zeitler, Bryan,

Guschin, Dmitry Y,

Faraji, Farhoud M,

Urnov, Fyodor D,

Harland, Richard M,

Rebar, Edward J,

Young, John J

2011

Overview

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:

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Publisher

National Academy of Sciences,National Acad Sciences

Subject

adults

/ Alleles

/ Amphibians

/ Animals

/ Animals, Genetically Modified

/ Anura

/ Binding sites