The use of TALENs for nonhomologous end joining mutagenesis in silkworm and fruitfly
The use of TALENs for nonhomologous end joining mutagenesis in silkworm and fruitfly
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DOI：
10.1016/j.ymeth.2014.02.014
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发表时间：
2014-08-15
期刊：
影响因子：
4.8
通讯作者：
Zurovec, Michal
中科院分区：
文献类型：
--
作者：
Takasu, Yoko;Tamura, Toshiki;Zurovec, Michal
转录激活因子样效应物核酸酶（TALEN）是定制的酶，其被设计为在所需位置切割双链DNA。DNA断裂通过易错非同源末端连接（NHEJ）途径或通过需要同源DNA作为修复模板的同源重组来修复。TALEN用于在包括昆虫在内的广泛范围的生物体中进行位点特异性诱变。我们将在这里描述一种简单的基于TALEN的突变方案，适合在家蚕和黑腹果蝇中产生种系突变。该方案包括特定TAL模块的组装、TALEN RNA的体外合成、卵子显微注射和使用PCR分析的突变检测。我们的方法允许高频率诱导NHEJ突变，这通常允许接收已经在G（1）中的纯合突变体。(C)2014爱思唯尔公司All rights reserved.
Transcription activator-like effector nucleases (TALENs) are custom-made enzymes designed to cut double-stranded DNA at desired locations. The DNA breaks are repaired either by error-prone non-homologous end-joining (NHEJ) pathway or via homologous recombination requiring homologous DNA as a template for the repair. TALENs are used for site-specific mutagenesis in an extended range of organisms including insects. We will describe here a simple TALEN-based mutagenesis protocol suitable for the generation of germline mutations in Bombyx mori and Drosophila melanogaster. The protocol includes assembly of specific TAL modules, in vitro synthesis of TALEN RNAs, egg microinjection and mutation detection using PCR analysis. Our procedure allows a high frequency induction of NHEJ mutations, which often allows the reception of homozygous mutants already in the G(1). (C) 2014 Elsevier Inc. All rights reserved.