Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University , 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526 , Japan.
Biol Open. 2013 Mar 4;2(5):448-52. doi: 10.1242/bio.20133855. Print 2013 May 15.
Recently, gene editing with transcription activator-like effector nucleases (TALENs) has been used in the life sciences. TALENs can be easily customized to recognize a specific DNA sequence and efficiently introduce double-strand breaks at the targeted genomic locus. Subsequent non-homologous end-joining repair leads to targeted gene disruption by base insertion, deletion, or both. Here, to readily evaluate the efficacy of TALENs in Xenopus laevis embryos, we performed the targeted gene disruption of tyrosinase (tyr) and pax6 genes that are involved in pigmentation and eye formation, respectively. We constructed TALENs targeting tyr and pax6 and injected their mRNAs into fertilized eggs at the one-cell stage. Expectedly, introduction of tyr TALEN mRNA resulted in drastic loss of pigmentation with high efficiency. Similarly, for pax6, TALENs led to deformed eyes in the injected embryos. We confirmed mutations of the target alleles by restriction enzyme digestion and sequence analyses of genomic PCR products. Surprisingly, not only biallelic but also paralogous, gene disruption was observed. Our results demonstrate that targeted gene disruption by TALENs provides a method comparable to antisense morpholinos in analyzing gene function in Xenopus F0 embryos, but also applies beyond embryogenesis to any life stage.
最近，转录激活因子样效应物核酸酶（TALEN）的基因编辑技术已被应用于生命科学领域。TALEN 可以很容易地被定制以识别特定的 DNA 序列，并在靶向基因组位点有效地引入双链断裂。随后的非同源末端连接修复导致靶向基因的碱基插入、缺失或同时发生的破坏。在这里，为了方便评估 TALEN 在非洲爪蟾胚胎中的功效，我们对分别参与色素形成和眼睛形成的酪氨酸酶（tyr）和 pax6 基因进行了靶向基因敲除。我们构建了靶向 tyr 和 pax6 的 TALEN，并将它们的 mRNA 在受精卵的单细胞期注射入胚胎。预期的是，tyr TALEN mRNA 的引入导致高效的严重色素损失。同样，对于 pax6，TALEN 导致注射胚胎的眼睛畸形。我们通过限制性内切酶消化和基因组 PCR 产物的序列分析来确认靶等位基因的突变。令人惊讶的是，不仅观察到了等位基因的双等位基因敲除，而且还观察到了同源基因的敲除。我们的结果表明，TALEN 介导的靶向基因敲除提供了一种与反义 morpholino 类似的方法，可用于分析非洲爪蟾 F0 胚胎中的基因功能，但也适用于胚胎发生以外的任何生命阶段。
