Establishment of an Efficient Immortalization Strategy Using HMEJ-Based bTERT Insertion for Bovine Cells.

Yong Zhang,Ying Guo,Zihan Zhang,Zhuo Han,Xin Liu,Yuanpeng Gao

doi:10.3390/ijms222212540

Abstract

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.

Highlights

Immortalized cell lines play an important role in the functional gene research and cell signaling pathway of cattle [1,2,3,4]
We revealed that the endogenous hindrance of this strategy in promoting the immortalization of bovine bone marrow-derived macrophages was mainly reflected in the blockage of nuclear translocation and inhibition of enzymatic activity of bovine TERT (bTERT) regulated by posttranslational modification
These results demonstrated that bTERT could replace the role of human telomerase reverse transcriptase (hTERT) in promoting cell survival by maintaining the telomerase activity and relative telomere length of cells due to the high conservation with hTERT in protein sequence, structure, and function. bTERT is more suitable for the immortalization of bovine cells than hTERT

Summary

Introduction

Immortalized cell lines play an important role in the functional gene research and cell signaling pathway of cattle [1,2,3,4]. Given the lack of bovine immortalized cell lines, most related studies on cattle only use human or mouse cell lines as model cells, which may hinder subsequent research and application [5,6]. The traditional immortalization strategies for bovine cells generally introduce human telomerase reverse transcriptase (hTERT) genes, oncogenes, or viral genomes into primary cells via transient transfection or a virus-mediated delivery system and construct long-term cultured cell lines in vitro [7]. There are some limitations and risks in the subsequent use of cell lines generated via these methods, such as the inefficiency of the immortalized cell lines caused by the short expression time of the immortalization factor via transient transfection, as well as the instability of the cell genome because of the integration of random exogenous immortalization factors through virus-mediated delivery [8,9,10,11,12,13]. An efficient and manipulated immortalized strategy exclusive for bovine cells is necessary

Methods

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Discussion

Conclusion