Targeted mutagenesis in the silkworm Bombyx mori using zinc finger nuclease mRNA injection
Targeted mutagenesis in the silkworm Bombyx mori using zinc finger nuclease mRNA injection
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DOI：
10.1016/j.ibmb.2010.07.012
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发表时间：
2010-10-01
影响因子：
3.8
通讯作者：
Zurovec, Michal
中科院分区：
文献类型：
--
作者：
Takasu, Yoko;Kobayashi, Isao;Zurovec, Michal
靶向诱变是功能基因分析的关键方法之一。一种简化的基因靶向变体，将定制的锌指核酸酶（ZFN） mrna直接微量注射到果蝇胚胎中。为了评估该方法在其他昆虫基因靶向中的适用性，我们诱变了家蚕表皮颜色标记基因bmbro2，该基因控制了家蚕幼虫表皮尿酸颗粒的形成。我们的研究结果表明，注射ZFN mRNA可以通过非同源末端连接（NHEJ）有效地诱导靶基因的体细胞和种系突变。zfn诱导的NHEJ突变缺乏末端填充和钝连接产物，主要包括7bp或更长长度的缺失，以及单核苷酸插入。这些观察结果表明，B. moti双链断裂修复系统依赖于微同源性，而不是典型的连接酶iv依赖机制。G(1)中种系突变的频率足以用于基因靶向，仅依靠基于分子方法的筛选。(C) 2010 Elsevier Ltd.版权所有。
Targeted mutagenesis is one of the key methods for functional gene analysis. A simplified variant of gene targeting uses direct microinjection of custom-designed Zinc Finger Nuclease (ZFN) mRNAs into Drosophila embryos. To evaluate the applicability of this method to gene targeting in another insect, we mutagenized the Bombyx mori epidermal color marker gene BmBLOS2, which controls the formation of uric acid granules in the larval epidermis. Our results revealed that ZFN mRNA injection is effective to induce somatic, as well as germline, mutations in a targeted gene by non-homologous end joining (NHEJ). The ZFN-induced NHEJ mutations lack end-filling and blunt ligation products, and include mainly 7 bp or longer deletions, as well as single nucleotide insertions. These observations suggest that the B. moti double-strand break repair system relies on microhomologies rather than on a canonical ligase IV-dependent mechanism. The frequency of germline mutants in G(1) was sufficient to be used for gene targeting relying on a screen based solely on molecular methods. (C) 2010 Elsevier Ltd. All rights reserved.