TALEN-mediated homologous recombination in Daphnia magna.
TALEN-mediated homologous recombination in Daphnia magna.
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DOI：
10.1038/srep18312
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发表时间：
2015-12-17
影响因子：
4.6
通讯作者：
Watanabe H
中科院分区：
文献类型：
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作者：
Nakanishi T;Kato Y;Matsuura T;Watanabe H
转录激活剂样效应核酸酶 (TALEN) 提供了多种工具来改造各种生物体中的内源基因组位点。我们使用 TALEN 在甲壳动物大型水蚤中建立了基于同源重组 (HR) 的基因敲入，这是一种生态和毒理学基因组学模型。我们构建了 TALEN，并设计了 67bp 供体插入片段，针对显示眼睛畸形的无眼突变体中的点缺失。将 TALEN mRNA 与供体 DNA 共同注射到卵子中，导致供体插入物精确整合到种系中，从而恢复了后代的眼睛畸形。通过使用与靶标具有 1.5kb 和 80nt 同源性的质粒和单链寡 DNA，种系中 HR 事件的频率为 2%。参与非同源末端连接修复的连接酶 4 的缺陷不会提高 HR 效率。我们的数据代表了 D. magna 中 TALEN 的高效基于 HR 的敲入，这是了解水蚤基因功能的有前途的工具。
Transcription Activator-Like Effector Nucleases (TALENs) offer versatile tools to engineer endogenous genomic loci in various organisms. We established a homologous recombination (HR)-based knock-in using TALEN in the crustacean Daphnia magna, a model for ecological and toxicological genomics. We constructed TALENs and designed the 67 bp donor insert targeting a point deletion in the eyeless mutant that shows eye deformities. Co-injection of the TALEN mRNA with donor DNA into eggs led to the precise integration of the donor insert in the germ line, which recovered eye deformities in offspring. The frequency of HR events in the germ line was 2% by using both plasmid and single strand oligo DNA with 1.5 kb and 80 nt homology to the target. Deficiency of ligase 4 involved in non-homologous end joining repair did not increase the HR efficiency. Our data represent efficient HR-based knock-in by TALENs in D. magna, which is a promising tool to understand Daphnia gene functions.