Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, 630-0192, Japan.
Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Sci Rep. 2021 Apr 15;11(1):8297. doi: 10.1038/s41598-021-87751-5.
E26 avian leukemia oncogene 2, 3' domain (Ets2) has been implicated in various biological processes. An Ets2 mutant model (Ets2), which lacks the DNA-binding domain, was previously reported to exhibit embryonic lethality caused by a trophoblast abnormality. This phenotype could be rescued by tetraploid complementation, resulting in pups with wavy hair and curly whiskers. Here, we generated new Ets2 mutant models with a frame-shift mutation in exon 8 using the CRISPR/Cas9 method. Homozygous mutants could not be obtained by natural mating as embryonic development stopped before E8.5, as previously reported. When we rescued them by tetraploid complementation, these mice did not exhibit wavy hair or curly whisker phenotypes. Our newly generated mice exhibited exon 8 skipping, which led to in-frame mutant mRNA expression in the skin and thymus but not in E7.5 Ets2 embryos. This exon 8-skipped Ets2 mRNA was translated into protein, suggesting that this Ets2 mutant protein complemented the Ets2 function in the skin. Our data implies that novel splicing variants incidentally generated after genome editing may complicate the phenotypic analysis but may also give insight into the new mechanisms related to biological gene functions.
E26 禽白血病致癌基因 2，3' 结构域（Ets2）参与了多种生物学过程。先前报道的一种缺乏 DNA 结合结构域的 Ets2 突变模型（Ets2）表现出由滋养层异常引起的胚胎致死性。这种表型可以通过四倍体互补来挽救，导致毛发卷曲、胡须卷曲的幼仔。在这里，我们使用 CRISPR/Cas9 方法生成了新的 Ets2 突变模型，其在 8 号外显子中发生了移码突变。与先前报道的一样，由于胚胎发育在 E8.5 之前停止，自然交配不能获得纯合突变体。当我们通过四倍体互补来拯救它们时，这些小鼠没有表现出卷曲的毛发或卷曲的胡须表型。我们新生成的小鼠出现了 8 号外显子跳跃，导致皮肤和胸腺中有框突变 mRNA 的表达，但在 E7.5 Ets2 胚胎中没有。这种 8 号外显子跳跃的 Ets2 mRNA 被翻译成蛋白质，表明这种 Ets2 突变蛋白在皮肤中补充了 Ets2 的功能。我们的数据表明，基因组编辑后偶然产生的新剪接变体可能会使表型分析复杂化，但也可能深入了解与生物学基因功能相关的新机制。
