IBSAL (Instituto de Investigación Biomédica de Salamanca), Salamanca, Spain.
Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain.
Mol Biol Rep. 2020 Feb;47(2):1381-1391. doi: 10.1007/s11033-019-05214-7. Epub 2019 Dec 12.
The Nomo1 gene mediates a wide range of biological processes of importance in embryonic development. Accordingly, constitutive perturbation of Nomo1 function may result in myriad developmental defects that trigger embryonic lethality. To extend our understanding of Nomo1 function in postnatal stages and in a tissue-specific manner, we generated a conditional knockout mouse model of Nomo1. To achieve this, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology in C57Bl/6J mouse zygotes to generate a new mouse model in which exon 3 of the Nomo1 gene is specifically flanked (or floxed) by LoxP sites (Nomo1). Nomo1 mouse embryonic fibroblasts were transduced with a Cre adenovirus and efficiently recombined between LoxP sites. Genomic and expression studies in Nomo1-transduced MEFs demonstrated that the Nomo1 exon 3 is ablated. Western blot assay showed that no protein or early truncated protein is produced. In vivo assay crossing Nomo1 mouse with a Msi1-CRE transgenic mouse corroborated the previous findings and it showed Nomo1 exon 3 deletion at msi1+ cell compartment. This short technical report demonstrates that CRISPR/Cas9 technology is a simple and easy method for creating conditional mouse models. The Nomo1 mouse will be useful to researchers who wish to explore the role of Nomo1 in any developmental stage or in a tissue-specific manner.
Nomo1 基因介导了胚胎发育中许多重要的生物学过程。因此，Nomo1 功能的组成性干扰可能导致无数的发育缺陷，从而引发胚胎致死。为了更深入地了解 Nomo1 在出生后的阶段以及组织特异性方面的功能，我们构建了 Nomo1 的条件性敲除小鼠模型。为此，我们使用了 C57Bl/6J 小鼠受精卵中的规律成簇间隔短回文重复序列 (CRISPR)/Cas9 技术，在 Nomo1 基因的第 3 外显子两侧产生了 LoxP 位点 (Nomo1)。Nomo1 小鼠胚胎成纤维细胞被 Cre 腺病毒转导，并在 LoxP 位点之间有效地重组。在 Nomo1 转导的 MEFs 中的基因组和表达研究表明，Nomo1 外显子 3 缺失。Western blot 检测表明，没有产生蛋白或早期截断蛋白。体内实验将 Nomo1 小鼠与 Msi1-CRE 转基因小鼠杂交，证实了上述发现，并显示了 msi1+细胞区室中 Nomo1 外显子 3 的缺失。这个简短的技术报告表明，CRISPR/Cas9 技术是一种创建条件性小鼠模型的简单方法。Nomo1 小鼠将对希望探索 Nomo1 在任何发育阶段或组织特异性方面作用的研究人员非常有用。
