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Targeted mutagenesis in the sea urchin embryo using zinc‐finger nucleases
Author(s) -
Ochiai Hiroshi,
Fujita Kazumasa,
Suzuki Kenichi,
Nishikawa Masatoshi,
Shibata Tatsuo,
Sakamoto Naoaki,
Yamamoto Takashi
Publication year - 2010
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1111/j.1365-2443.2010.01425.x
Subject(s) - biology , zinc finger nuclease , sea urchin , zinc finger , mutagenesis , embryo , microbiology and biotechnology , genetics , computational biology , mutation , gene , transcription factor
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.