Due to their cost effectiveness, ease of use, and unlimited supply, immortalized cell lines are used in place of primary cells for a wide range of research purposes, including gene function studies, CRISPR-based gene editing, drug metabolism tests, and vaccine or therapeutic protein production. Although immortalized cell lines have been established for a range of animal species, there is still a need to develop such cell lines for wild species. The zebra finch, which is used widely as a model species to study the neurobiological basis of human speech disorders, has been employed in several functional studies involving gene knockdown or the introduction of exogenous transgenes in vivo; however, the lack of an immortalized zebra finch cell line has hampered precise genome editing studies. Here, we established an immortalized cell line by a single genetic event, expression of the c-MYC oncogene, in zebra finch embryonic fibroblasts and examined its potential suitability for gene targeting investigations. Retroviral vector-mediated transduction of c-MYC was used to immortalize zebra finch primary fibroblasts; the transformed cells proliferated stably over several passages, resulting in the expression of chondrocyte-specific genes. The transfection efficiency of the immortalized cells was much higher than that of the primary cells. Targeted knockout of the SOX9 gene, which plays a role in the differentiation of mesenchymal progenitor cells into chondrocytes, was conducted in vitro and both apoptosis and decreased expression levels of chondrogenic marker genes were observed in edited cells. The c-MYC induced immortalized chondrocyte-like cell line described here broadens the available options for establishing zebra finch cell lines, paves the way for in-depth biological researches, and provides convenient approaches for biotechnology studies, particularly genomic modification research.

中文翻译：

---

通过 c-MYC 表达永生化的斑胸草雀细胞系基因组修饰的软骨细胞样细胞的产生和表征

由于其成本效益、易用性和无限供应，永生化细胞系代替原代细胞用于广泛的研究目的，包括基因功能研究、基于 CRISPR 的基因编辑、药物代谢测试和疫苗或治疗性蛋白质生产。尽管已经为一系列动物物种建立了永生化细胞系，但仍然需要为野生物种开发此类细胞系。斑胸草雀被广泛用作研究人类语言障碍的神经生物学基础的模型物种，已被用于涉及基因敲低或体内外源转基因引入的多项功能研究。然而，缺乏永生化斑胸草雀细胞系阻碍了精确的基因组编辑研究。这里，我们通过单一遗传事件，在斑胸草雀胚胎成纤维细胞中表达 c-MYC 癌基因，建立了永生化细胞系，并检查了其对基因靶向研究的潜在适用性。逆转录病毒载体介导的 c-MYC 转导用于使斑胸草雀原代成纤维细胞永生化；转化的细胞在几代后稳定增殖，导致软骨细胞特异性基因的表达。永生化细胞的转染效率远高于原代细胞。在体外进行了 SOX9 基因的靶向敲除，该基因在间充质祖细胞分化为软骨细胞中起作用，并且在编辑的细胞中观察到细胞凋亡和软骨形成标记基因的表达水平降低。