Targeted mutagenesis in the sea urchin embryo using zinc-finger nucleases
Targeted mutagenesis in the sea urchin embryo using zinc-finger nucleases
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DOI：
10.1111/j.1365-2443.2010.01425.x
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发表时间：
2010-08-01
期刊：
影响因子：
2.1
通讯作者：
Yamamoto, Takashi
中科院分区：
文献类型：
--
作者：
Ochiai, Hiroshi;Fujita, Kazumasa;Yamamoto, Takashi
我们发现，工程化的锌指核酸酶（ZFN），它包括一个锌指DNA结合阵列和限制性内切酶FokI的核酸酶结构域，可以在海胆胚胎的特定基因组位点引入突变。使用细菌单杂交筛选与锌指随机库和单链退火试验在培养的细胞中，ZFN靶向海胆Hemicentrotus pulcherrimus同源的HesC（HpHesC）有效地选择。与在注射了针对HpHesC的反义吗啉代寡核苷酸的胚胎中观察到的表型一致，在注射了一对HpHesC ZFN mRNA的胚胎中观察到原代间充质细胞群的增加。此外，突变的序列分析表明，在注射HpHesC ZFN mRNA的胚胎中，HpHesC靶位点发生缺失和插入。这些结果表明，使用ZFN的靶向基因破坏是可行的海胆胚胎。
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.