RIKEN BioResource Research Center, Ibaraki, Japan.
Department of Disease Model, Research Institute of Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan.
Biol Reprod. 2021 Jan 4;104(1):223-233. doi: 10.1093/biolre/ioaa176.
Conditional knockout (cKO) mice have contributed greatly to understanding the tissue- or stage-specific functions of genes in vivo. However, the current cKO method requires considerable time and effort because of the need to generate two gene-modified mouse strains (Cre transgenic and loxP knockin) for crossing. Here, we examined whether we could analyze the germ cell-related functions of embryonic lethal genes in F0 chimeric mice by restricting the origin of germ cells to mutant embryonic stem cells (ESCs). We confirmed that the full ESC origin of spermatozoa in fertile chimeric mice was achieved by the CRISPR/Cas9 system using three guide RNAs targeting Nanos3, which induced germ cell depletion in the host blastocyst-derived tissues. Among these fertile chimeric mice, those from male ESCs with a Dnmt3b mutation, which normally causes embryo death, also produced F1 mice derived exclusively from the mutant ESCs. Thus, our new chimeric strategy readily revealed that Dnmt3b is dispensable for male germ cell development, in agreement with a previous cKO study. Our new approach enables us to analyze the germ cell functions of embryonic lethal genes in the F0 generation without using the current cKO method.
条件性基因敲除（cKO）小鼠在体内研究基因的组织或阶段特异性功能方面做出了巨大贡献。然而，由于需要生成两种基因修饰的小鼠品系（Cre 转基因和 loxP 敲入）进行杂交，目前的 cKO 方法需要相当多的时间和精力。在这里，我们研究了是否可以通过将生殖细胞的起源限制在突变的胚胎干细胞（ESCs）中来分析 F0 嵌合小鼠中与生殖细胞相关的胚胎致死基因的功能。我们使用靶向 Nanos3 的三个向导 RNA 证实了 CRISPR/Cas9 系统能够实现精子的完整 ESC 起源，该系统在宿主囊胚衍生组织中诱导了生殖细胞耗竭。在这些可育嵌合小鼠中，那些源自正常导致胚胎死亡的 Dnmt3b 突变的雄性 ESCs 也产生了仅源自突变 ESCs 的 F1 小鼠。因此，我们的新嵌合策略很容易表明 Dnmt3b 对于雄性生殖细胞发育是可有可无的，这与之前的 cKO 研究一致。我们的新方法使我们能够在不使用当前 cKO 方法的情况下在 F0 代中分析胚胎致死基因的生殖细胞功能。
