利用转录激活样效应因子核酸酶（TALENs）在半变态昆虫双斑蟋（Gryllus bimaculatus）中通过靶向诱变进行基因敲除

GeneKnockout by Targeted Mutagenesis in a Hemimetabolous Insect, the Two-Spotted Cricket Gryllus bimaculatus, using TALENs.

作者信息

Watanabe Takahito, Noji Sumihare, Mito Taro

机构信息

Department of Life Systems, Institute of Technology and Science, The University of Tokushima, 2-1 Minami-Jyosanjima-cho, Tokushima, 770-0815, Japan.

Center for Collaboration among Agriculture, Industry and Commerce, The University of Tokushima, 2-1 Minami-Jyosanjima-cho, Tokushima, 770-0815, Japan.

出版信息

Methods Mol Biol. 2016;1338:143-55. doi: 10.1007/978-1-4939-2932-0_12.

DOI:10.1007/978-1-4939-2932-0_12

PMID:26443220

Abstract

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.

摘要

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）是半变态昆虫的新兴模型，这是基于基于RNA干扰（RNAi）的基因功能分析和转基因技术的成功。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是能够在特定目标序列中诱导DNA双链断裂的人工核酸酶。在这里，我们描述了一种在双斑蟋中基于TALEN的基因敲除方案，包括通过突变检测分析的突变体选择方案，用于产生纯合敲除生物体。