Division of Embryology and Genetics, Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Higashihiroshima 739-8526, Japan.
Biol Open. 2013 Dec 15;2(12):1364-70. doi: 10.1242/bio.20136676.
Transcription activator-like effector nucleases (TALENs) are facile and potent tools used to modify a gene of interest for targeted gene knockout. TALENs consist of an N-terminal domain, a DNA-binding domain, and a C-terminal domain, which are derived from a transcription activator-like effector, and the non-specific nuclease domain of FokI. Using Xenopus tropicalis (X. tropicalis), we compared the toxicities and somatic mutation activities of four TALEN architectures in a side-by-side manner: a basic TALEN, a scaffold with the same truncated N- and C-terminal domains as GoldyTALEN, a scaffold with the truncated N- and C-terminal domains and an obligate heterodimeric nuclease domain, and a scaffold with the truncated N- and C-terminal domains and an obligate heterodimeric Sharkey nuclease domain. The strongest phenotype and targeted somatic gene mutation were induced by the injection of TALEN mRNAs containing the truncated N- and C-terminal domains and an obligate heterodimeric nuclease domain. The obligate heterodimeric TALENs exhibited reduced toxicity compared to the homodimeric TALENs, and the homodimeric GoldyTALEN-type scaffold showed both a high activity of somatic gene modification and high toxicity. The Sharkey mutation in the heterodimeric nuclease domain reduced the TALEN-mediated somatic mutagenesis.
转录激活样效应物核酸酶（TALENs）是一种易于使用且有效的工具，可用于修饰感兴趣的基因以进行靶向基因敲除。TALENs 由一个 N 端结构域、一个 DNA 结合结构域和一个 C 端结构域组成，它们分别来源于转录激活样效应物和 FokI 的非特异性核酸酶结构域。我们使用非洲爪蟾（X. tropicalis），以并排的方式比较了四种 TALEN 结构的毒性和体细胞突变活性：一种基本 TALEN、一种 GoldyTALEN 具有相同截断 N 和 C 端结构域的支架、一种具有截断 N 和 C 端结构域和必需异源二聚体核酸酶结构域的支架，以及一种具有截断 N 和 C 端结构域和必需异源二聚体 Sharkey 核酸酶结构域的支架。含有截断 N 和 C 端结构域和必需异源二聚体核酸酶结构域的 TALEN mRNA 的注射诱导了最强的表型和靶向体细胞基因突变。与同源二聚体 TALEN 相比，必需异源二聚体 TALEN 的毒性降低，而同源二聚体 GoldyTALEN 型支架则显示出高体细胞基因修饰活性和高毒性。异源二聚体核酸酶结构域中的 Sharkey 突变降低了 TALEN 介导的体细胞诱变。
