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Construction and Characterization of Immortalized Fibroblast Cell Line from Bactrian Camel

Immortalized cell lines with many advantages are widely used in various experimental contexts by many different labs. However, the absence of available cell lines poses difficulties for research in some species, such as camels. To establish an immortalized Bactrian camel fibroblast (iBCF) cell line...

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Autores principales: Yan, Meilin, Yong, Fang, Ji, Wangye, Zhang, Lili, Zhao, Shuqin, Gao, Yuan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10302944/
https://www.ncbi.nlm.nih.gov/pubmed/37374120
http://dx.doi.org/10.3390/life13061337
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author Yan, Meilin
Yong, Fang
Ji, Wangye
Zhang, Lili
Zhao, Shuqin
Gao, Yuan
author_facet Yan, Meilin
Yong, Fang
Ji, Wangye
Zhang, Lili
Zhao, Shuqin
Gao, Yuan
author_sort Yan, Meilin
collection PubMed
description Immortalized cell lines with many advantages are widely used in various experimental contexts by many different labs. However, the absence of available cell lines poses difficulties for research in some species, such as camels. To establish an immortalized Bactrian camel fibroblast (iBCF) cell line and understand its biological characteristics, primary fibroblast cells from Bactrian camels were isolated and purified using enzymatic digestion in this study, and telomerase reverse transcriptase (hTERT) vectors were introduced into primary BCF (pBCF) for continuous passage to 80 generations after screening with G418. The cell morphology of different generations was examined under a microscope. Cell cycle and viability were evaluated by flow cytometry and CCK-8 assay, respectively. Cellular genes expression was monitored by qPCR, immunofluorescence, and Western blot, respectively. Chromosomes were determined by karyotyping. The results showed that like most other cells, both pBCF and iBCF were sensitive to nutrient concentrations and adapted to culture in the medium with 4.5 g/L glucose and 10% fetal bovine serum (FBS) concentration. hTERT gene was introduced and stably expressed in iBCF cells, which promoted BCF cell immortalization. The fibroblast specific marker vimentin (VIM) is expressed in both pBCF and iBCF, but epithelial marker cytokeratin18 (CK18) expression is weak in BCF cells. Proliferation and viability detection showed that hTERT-induced iBCF exhibits faster growth rates and higher viability than pBCF. Karyotyping showed that iBCF maintained the same number and morphology of chromosomes as the pBCF. This study demonstrated that we have successfully constructed an immortalized Bactrian camel fibroblast cell line, which was named BCF23. The establishment of the BCF23 cell line provides a foundation for expanding camel-related research.
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spelling pubmed-103029442023-06-29 Construction and Characterization of Immortalized Fibroblast Cell Line from Bactrian Camel Yan, Meilin Yong, Fang Ji, Wangye Zhang, Lili Zhao, Shuqin Gao, Yuan Life (Basel) Article Immortalized cell lines with many advantages are widely used in various experimental contexts by many different labs. However, the absence of available cell lines poses difficulties for research in some species, such as camels. To establish an immortalized Bactrian camel fibroblast (iBCF) cell line and understand its biological characteristics, primary fibroblast cells from Bactrian camels were isolated and purified using enzymatic digestion in this study, and telomerase reverse transcriptase (hTERT) vectors were introduced into primary BCF (pBCF) for continuous passage to 80 generations after screening with G418. The cell morphology of different generations was examined under a microscope. Cell cycle and viability were evaluated by flow cytometry and CCK-8 assay, respectively. Cellular genes expression was monitored by qPCR, immunofluorescence, and Western blot, respectively. Chromosomes were determined by karyotyping. The results showed that like most other cells, both pBCF and iBCF were sensitive to nutrient concentrations and adapted to culture in the medium with 4.5 g/L glucose and 10% fetal bovine serum (FBS) concentration. hTERT gene was introduced and stably expressed in iBCF cells, which promoted BCF cell immortalization. The fibroblast specific marker vimentin (VIM) is expressed in both pBCF and iBCF, but epithelial marker cytokeratin18 (CK18) expression is weak in BCF cells. Proliferation and viability detection showed that hTERT-induced iBCF exhibits faster growth rates and higher viability than pBCF. Karyotyping showed that iBCF maintained the same number and morphology of chromosomes as the pBCF. This study demonstrated that we have successfully constructed an immortalized Bactrian camel fibroblast cell line, which was named BCF23. The establishment of the BCF23 cell line provides a foundation for expanding camel-related research. MDPI 2023-06-07 /pmc/articles/PMC10302944/ /pubmed/37374120 http://dx.doi.org/10.3390/life13061337 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Yan, Meilin
Yong, Fang
Ji, Wangye
Zhang, Lili
Zhao, Shuqin
Gao, Yuan
Construction and Characterization of Immortalized Fibroblast Cell Line from Bactrian Camel
title Construction and Characterization of Immortalized Fibroblast Cell Line from Bactrian Camel
title_full Construction and Characterization of Immortalized Fibroblast Cell Line from Bactrian Camel
title_fullStr Construction and Characterization of Immortalized Fibroblast Cell Line from Bactrian Camel
title_full_unstemmed Construction and Characterization of Immortalized Fibroblast Cell Line from Bactrian Camel
title_short Construction and Characterization of Immortalized Fibroblast Cell Line from Bactrian Camel
title_sort construction and characterization of immortalized fibroblast cell line from bactrian camel
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10302944/
https://www.ncbi.nlm.nih.gov/pubmed/37374120
http://dx.doi.org/10.3390/life13061337
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