title: Targeted mutagenesis in the sea urchin embryo using zinc-finger nucleases.
abstract: We showed that engineered zinc-finger nucleases (ZFNs), which consist of a zinc-finger DNA-binding array and a nuclease domain of the restriction enzyme FokI, can introduce mutations at a specific genomic site in the sea urchin embryo. Using bacterial one-hybrid screening with zinc-finger randomized libraries and a single-strand annealing assay in cultured cells, ZFNs targeting the sea urchin Hemicentrotus pulcherrimus homologue of HesC (HpHesC) were efficiently selected. Consistent with the phenotype observed in embryos injected with an antisense morpholino oligonucleotide against HpHesC, an increase in the primary mesenchyme cell population was observed in embryos injected with a pair of HpHesC ZFN mRNAs. In addition, sequence analysis of the mutations showed that deletions and insertions occurred at the HpHesC target site in the embryos injected with the HpHesC ZFN mRNAs. These results suggest that targeted gene disruption using ZFNs is feasible for the sea urchin embryo.
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ochiai
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Fujita
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Suzuki
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Nishikawa
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Shibata
authors.forename: Naoaki
authors.initials: N
authors.lastname: Sakamoto
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamamoto
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004720:Endonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D005818:Genetic Engineering; D047330:Hemicentrotus / Q000196:embryology* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics; D016335:Zinc Fingers
chemical_list: D012333:RNA, Messenger; D004720:Endonucleases