Establishment of an Efficient Immortalization Strategy Using HMEJ-Based b<i>TERT</i> Insertion for Bovine Cells

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...

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Autores principales:	Zihan Zhang, Zhuo Han, Ying Guo, Xin Liu, Yuanpeng Gao, Yong Zhang
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	immortalization bovine telomerase reverse transcriptase (<i>TERT</i>) homology-mediated end joining (HMEJ) CRISPR/Cas9 macrophage Biology (General) QH301-705.5 Chemistry QD1-999
Acceso en línea:	https://doaj.org/article/20ef9ad7fbff40d2a5ff927cc554adc1
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spelling	oai:doaj.org-article:20ef9ad7fbff40d2a5ff927cc554adc12021-11-25T17:57:38ZEstablishment of an Efficient Immortalization Strategy Using HMEJ-Based b<i>TERT</i> Insertion for Bovine Cells10.3390/ijms2222125401422-00671661-6596https://doaj.org/article/20ef9ad7fbff40d2a5ff927cc554adc12021-11-01T00:00:00Zhttps://www.mdpi.com/1422-0067/22/22/12540https://doaj.org/toc/1661-6596https://doaj.org/toc/1422-0067Immortalized 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.Zihan ZhangZhuo HanYing GuoXin LiuYuanpeng GaoYong ZhangMDPI AGarticleimmortalizationbovinetelomerase reverse transcriptase (<i>TERT</i>)homology-mediated end joining (HMEJ)CRISPR/Cas9macrophageBiology (General)QH301-705.5ChemistryQD1-999ENInternational Journal of Molecular Sciences, Vol 22, Iss 12540, p 12540 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	immortalization bovine telomerase reverse transcriptase (<i>TERT</i>) homology-mediated end joining (HMEJ) CRISPR/Cas9 macrophage Biology (General) QH301-705.5 Chemistry QD1-999
spellingShingle	immortalization bovine telomerase reverse transcriptase (<i>TERT</i>) homology-mediated end joining (HMEJ) CRISPR/Cas9 macrophage Biology (General) QH301-705.5 Chemistry QD1-999 Zihan Zhang Zhuo Han Ying Guo Xin Liu Yuanpeng Gao Yong Zhang Establishment of an Efficient Immortalization Strategy Using HMEJ-Based b<i>TERT</i> Insertion for Bovine Cells
description	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.
format	article
author	Zihan Zhang Zhuo Han Ying Guo Xin Liu Yuanpeng Gao Yong Zhang
author_facet	Zihan Zhang Zhuo Han Ying Guo Xin Liu Yuanpeng Gao Yong Zhang
author_sort	Zihan Zhang
title	Establishment of an Efficient Immortalization Strategy Using HMEJ-Based b<i>TERT</i> Insertion for Bovine Cells
title_short	Establishment of an Efficient Immortalization Strategy Using HMEJ-Based b<i>TERT</i> Insertion for Bovine Cells
title_full	Establishment of an Efficient Immortalization Strategy Using HMEJ-Based b<i>TERT</i> Insertion for Bovine Cells
title_fullStr	Establishment of an Efficient Immortalization Strategy Using HMEJ-Based b<i>TERT</i> Insertion for Bovine Cells
title_full_unstemmed	Establishment of an Efficient Immortalization Strategy Using HMEJ-Based b<i>TERT</i> Insertion for Bovine Cells
title_sort	establishment of an efficient immortalization strategy using hmej-based b<i>tert</i> insertion for bovine cells
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/20ef9ad7fbff40d2a5ff927cc554adc1
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Establishment of an Efficient Immortalization Strategy Using HMEJ-Based b<i>TERT</i> Insertion for Bovine Cells

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