College of Veterinary Medicine, Northwest A&F University, Xianyang 712100, China.
Key Laboratory of Animal Biotechnology, Ministry of Agriculture and Rural Affairs, Northwest A&F University, Xianyang 712100, China.
Int J Mol Sci. 2021 Nov 21;22(22):12540. doi: 10.3390/ijms222212540.
Immortalized cell lines have been used in a wide range of applications in research on immune disorders and cellular metabolic regulation due to the stability and uniformity of their cellular characteristics. At present, the investigation into molecular functions and signaling pathways within bovine cells remains largely limited by the lack of immortalized model cells. Current methods for immortalizing bovine cells are mainly restricted to the ectopic expression of human telomerase reverse transcriptase (hTERT) through transient transfection or virus-mediated delivery, which have defects in efficiency and reliability. In this study, we identified bovine TERT (bTERT) as a novel potent biofactor for immortalizing bovine cells with great advantages over hTERT, and established an efficient and easily manipulated strategy for the immortalization of bovine primary cells. Through the homology-mediated end-joining-based insertion of bTERT at the ROSA26 locus, we successfully generated immortalized bovine fetal fibroblast cell lines with stable characteristics. The observed limitation of this strategy in immortalizing bovine bone marrow-derived macrophages was attributed to the post-translational modification of bTERT, causing inhibited nuclear localization and depressed activity of bTERT in this terminally differentiated cell. In summary, we constructed an innovative method to achieve the high-quality immortalization of bovine primary cells, thereby expanding the prospects for the future application of immortalized bovine model cell lines.
永生化细胞系由于其细胞特征的稳定性和均一性，已被广泛应用于免疫紊乱和细胞代谢调控的研究中。目前，对牛细胞内分子功能和信号通路的研究在很大程度上受到缺乏永生化模型细胞的限制。目前牛细胞永生化的方法主要局限于通过瞬时转染或病毒介导的方式异位表达人端粒酶逆转录酶（hTERT），其效率和可靠性存在缺陷。在这项研究中，我们鉴定出牛端粒酶（bTERT）是一种新型的有效的牛细胞永生化生物因子，具有优于 hTERT 的优势，并建立了一种高效且易于操作的牛原代细胞永生化策略。通过同源介导的末端连接将 bTERT 插入 ROSA26 基因座，我们成功地生成了具有稳定特征的永生化牛胎儿成纤维细胞系。该策略在永生化牛骨髓来源巨噬细胞中的局限性归因于 bTERT 的翻译后修饰，导致 bTERT 在这个终末分化的细胞中核定位抑制和活性降低。总之，我们构建了一种创新的方法来实现牛原代细胞的高质量永生化，从而为永生化牛模型细胞系的未来应用前景提供了广阔的空间。
