Takasu Yoko, Tamura Toshiki, Sajwan Suresh, Kobayashi Isao, Zurovec Michal
National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.
Biology Centre of the ASCR, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic.
Methods. 2014 Aug 15;69(1):46-57. doi: 10.1016/j.ymeth.2014.02.014. Epub 2014 Feb 22.
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 G1.
转录激活样效应因子核酸酶（TALENs）是一种定制酶，旨在在所需位置切割双链DNA。DNA断裂可通过易出错的非同源末端连接（NHEJ）途径修复，或通过同源重组修复，同源重组需要同源DNA作为修复模板。TALENs可用于包括昆虫在内的多种生物体的位点特异性诱变。在此，我们将描述一种基于TALEN的简单诱变方案，适用于在家蚕和黑腹果蝇中产生生殖系突变。该方案包括特定TAL模块的组装、TALEN RNA的体外合成、卵显微注射以及使用PCR分析进行突变检测。我们的方法可高频诱导NHEJ突变，这通常使得在G1代就能获得纯合突变体。
