Watanabe Takahito, Noji Sumihare, Mito Taro
Department of Life Systems, Institute of Technology and Science, The University of Tokushima, 2-1 Minami-Jyosanjima-cho, Tokushima 770-0815, Japan.
Department of Life Systems, Institute of Technology and Science, The University of Tokushima, 2-1 Minami-Jyosanjima-cho, Tokushima 770-0815, Japan.
Methods. 2014 Aug 15;69(1):17-21. doi: 10.1016/j.ymeth.2014.05.006. Epub 2014 May 27.
Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically basal. These insects include many deleterious species. The cricket, Gryllus bimaculatus, is an emerging model for hemimetabolous insects, based on the success of RNA interference (RNAi)-based gene-functional analyses and transgenic technology. Taking advantage of genome-editing technologies in this species would greatly promote functional genomics studies. Genome editing using transcription activator-like effector nucleases (TALENs) has proven to be an effective method for site-specific genome manipulation in various species. TALENs are artificial nucleases that are capable of inducing DNA double-strand breaks into specified target sequences. Here, we describe a protocol for TALEN-based gene knockout in G. bimaculatus, including a mutant selection scheme via mutation detection assays, for generating homozygous knockout organisms.
半变态昆虫，即不完全变态昆虫，在系统发育上处于基部位置。这些昆虫包括许多有害物种。双斑蟋（Gryllus bimaculatus）是基于基于RNA干扰（RNAi）的基因功能分析和转基因技术的成功，而成为半变态昆虫的新兴模型。利用该物种的基因组编辑技术将极大地促进功能基因组学研究。使用转录激活样效应物核酸酶（TALENs）进行基因组编辑已被证明是在各种物种中进行位点特异性基因组操作的有效方法。TALENs是一种人工核酸酶，能够在特定的靶序列中诱导DNA双链断裂。在此，我们描述了一种在双斑蟋中基于TALENs的基因敲除方案，包括通过突变检测分析进行突变体选择的方案，用于产生纯合敲除生物体。
