title: Targeted gene disruption in the Xenopus tropicalis genome using designed TALE nucleases.
abstract: Transcription activator-like effector nucleases (TALENs) are attractive and powerful molecular tools for targeted gene disruption because of their simple design and quick assembly. To evaluate the utility of TALENs in genome editing in Xenopus tropicalis, we prepared nine pairs of TALENs for the tyrosinase, noggin and MMP-9TH genes. All of the TALENs had some activity in a single-strand annealing assay using a cultured frog cell line, suggesting double-stranded DNA cleavage activity by the TALENs at their target site. The injection of mRNAs encoding TALENs into fertilized X. tropicalis embryos resulted in Cel-1 cleavage of the PCR fragment containing the target site amplified from embryo genomic DNA, indicating that a mutation in the target gene had occurred during embryogenesis. These mutations were confirmed by the sequencing of clones derived from the PCR fragments of genomic DNA. Patches of vitiligo were observed in tadpoles raised from fertilized eggs that had been injected with mRNAs of TALENs for the tyrosinase gene. TALENs containing the repeat variable di-residue (RVD) NN appeared to show more activity than TALENs containing RVD NK, although both RVD NN and NK preferentially associate with a G nucleotide.
authors.affiliation: Division of Embryology and Genetics, Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Higashihiroshima 739-8526, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Nakajima
authors.forename: Yuya
authors.initials: Y
authors.lastname: Nakai
authors.forename: Morihiro
authors.initials: M
authors.lastname: Okada
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Yaoita
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D016678:Genome; D007814:Larva; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D014981:Xenopus / Q000235:genetics / Q000378:metabolism*
chemical_list: D003851:Deoxyribonucleases