Recently, gene-editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) technique has attempted to utilize fibroblasts of livestock animals for somatic cell nuclear transfer. In this study, we establish the procedure for preparing skin fibroblast clones whose genes were edited by the CRISPR/Cas9 technique. After isolating fibroblasts from earlobes of Japanese Black cattle, subsequent collagenase-digestion and extensive wash procedures enabled us to avoid contamination of fungi. Electroporation using NEPA21, rather than lipofection using commercially available liposome reagents, allowed us to perform more efficient transfection of plasmid constructs. Although bovine ear-derived fibroblasts were not able to proliferate in single cell cultures in Dulbecco's modified Eagle medium containing 10% fetal calf serum, supplementation with insulin-transferrin-selenium mixture, human recombinant epidermal growth factor, or human recombinant basic fibroblast growth factor promoted proliferation of the cells, even in a single cell culture. Taking advantage of our established protocol, we eventually obtained eight ear-derived fibroblast clones with a recessive mutation in the isoleucyl-tRNA synthetase gene corrected by the CRISPR/Cas9 technique.

中文翻译：

---

建立用于体细胞核移植的基因编辑牛耳来源成纤维细胞的制备方案的建立。

最近，使用簇状规则间隔的短回文重复序列（CRISPR）/ CRISPR相关蛋白9（Cas9）技术进行基因编辑已尝试利用牲畜的成纤维细胞进行体细胞核转移。在这项研究中，我们建立了制备皮肤成纤维细胞克隆的程序，该克隆的基因已通过CRISPR / Cas9技术进行了编辑。从日本黑牛的耳垂中分离出成纤维细胞后，随后的胶原酶消化和大量洗涤程序使我们避免了真菌的污染。使用NEPA21进行电穿孔，而不是使用市售脂质体试剂进行脂质转染，使我们能够更有效地转染质粒构建体。尽管牛耳来源的成纤维细胞不能在Dulbecco'的单细胞培养物中增殖 改良的Eagle培养基含有10％的胎牛血清，补充胰岛素-转铁蛋白-硒混合物，人重组表皮生长因子或人重组碱性成纤维细胞生长因子，即使在单细胞培养中也能促进细胞增殖。利用我们已建立的方案，我们最终获得了八个源自耳朵的成纤维细胞克隆，它们通过CRISPR / Cas9技术校正了异亮氨酸-tRNA合成酶基因中的隐性突变。