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Evaluating the use of a CpG free promoter for long-term recombinant protein expression stability in Chinese hamster ovary cells

BACKGROUND: Methylated CpG dinucleotides in promoters are associated with the loss of gene expression in recombinant Chinese hamster ovary (CHO) cells during large-scale commercial manufacturing. We evaluated a promoter devoid of CpG dinucleotides, CpGfree, in parallel with a similar CpG containing...

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Autores principales: Ho, Steven C. L., Koh, Esther Y. C., Soo, Benjamin P. C., Mariati, Chao, Sheng-Hao, Yang, Yuansheng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5070371/
https://www.ncbi.nlm.nih.gov/pubmed/27756290
http://dx.doi.org/10.1186/s12896-016-0300-y
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author Ho, Steven C. L.
Koh, Esther Y. C.
Soo, Benjamin P. C.
Mariati
Chao, Sheng-Hao
Yang, Yuansheng
author_facet Ho, Steven C. L.
Koh, Esther Y. C.
Soo, Benjamin P. C.
Mariati
Chao, Sheng-Hao
Yang, Yuansheng
author_sort Ho, Steven C. L.
collection PubMed
description BACKGROUND: Methylated CpG dinucleotides in promoters are associated with the loss of gene expression in recombinant Chinese hamster ovary (CHO) cells during large-scale commercial manufacturing. We evaluated a promoter devoid of CpG dinucleotides, CpGfree, in parallel with a similar CpG containing promoter, CpGrich, for their ability to maintain the expression of recombinant enhanced green fluorescent protein (EGFP) after 8 weeks of culturing. RESULTS: While the promoters gave similar transient expression levels, CpGfree clones had significantly higher average stable expression possibly due to increased resistance to early silencing during integration into the chromosome. A greater proportion of cells in clones generated using the CpGfree promoter were still expressing detectable levels of EGFP after 8 weeks but the relative expression levels measured at week 8 to those measured at week 0 did not improve compared to clones generated using the CpGrich promoter. Chromatin immunoprecipitation assays indicated that the repression of the CpGfree promoter was likely linked to histone deacetylation and methylation. Use of histone deacetylase inhibitors also managed to recover some of the lost expression. CONCLUSION: Using a promoter without CpG dinucleotides could mitigate the early gene silencing but did not improve longer-term expression stability as silencing due to histone modifications could still take place. The results presented here would aid in promoter selection and design for improved protein production in CHO and other mammalian cells.
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spelling pubmed-50703712016-10-24 Evaluating the use of a CpG free promoter for long-term recombinant protein expression stability in Chinese hamster ovary cells Ho, Steven C. L. Koh, Esther Y. C. Soo, Benjamin P. C. Mariati Chao, Sheng-Hao Yang, Yuansheng BMC Biotechnol Research Article BACKGROUND: Methylated CpG dinucleotides in promoters are associated with the loss of gene expression in recombinant Chinese hamster ovary (CHO) cells during large-scale commercial manufacturing. We evaluated a promoter devoid of CpG dinucleotides, CpGfree, in parallel with a similar CpG containing promoter, CpGrich, for their ability to maintain the expression of recombinant enhanced green fluorescent protein (EGFP) after 8 weeks of culturing. RESULTS: While the promoters gave similar transient expression levels, CpGfree clones had significantly higher average stable expression possibly due to increased resistance to early silencing during integration into the chromosome. A greater proportion of cells in clones generated using the CpGfree promoter were still expressing detectable levels of EGFP after 8 weeks but the relative expression levels measured at week 8 to those measured at week 0 did not improve compared to clones generated using the CpGrich promoter. Chromatin immunoprecipitation assays indicated that the repression of the CpGfree promoter was likely linked to histone deacetylation and methylation. Use of histone deacetylase inhibitors also managed to recover some of the lost expression. CONCLUSION: Using a promoter without CpG dinucleotides could mitigate the early gene silencing but did not improve longer-term expression stability as silencing due to histone modifications could still take place. The results presented here would aid in promoter selection and design for improved protein production in CHO and other mammalian cells. BioMed Central 2016-10-18 /pmc/articles/PMC5070371/ /pubmed/27756290 http://dx.doi.org/10.1186/s12896-016-0300-y Text en © The Author(s). 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Ho, Steven C. L.
Koh, Esther Y. C.
Soo, Benjamin P. C.
Mariati
Chao, Sheng-Hao
Yang, Yuansheng
Evaluating the use of a CpG free promoter for long-term recombinant protein expression stability in Chinese hamster ovary cells
title Evaluating the use of a CpG free promoter for long-term recombinant protein expression stability in Chinese hamster ovary cells
title_full Evaluating the use of a CpG free promoter for long-term recombinant protein expression stability in Chinese hamster ovary cells
title_fullStr Evaluating the use of a CpG free promoter for long-term recombinant protein expression stability in Chinese hamster ovary cells
title_full_unstemmed Evaluating the use of a CpG free promoter for long-term recombinant protein expression stability in Chinese hamster ovary cells
title_short Evaluating the use of a CpG free promoter for long-term recombinant protein expression stability in Chinese hamster ovary cells
title_sort evaluating the use of a cpg free promoter for long-term recombinant protein expression stability in chinese hamster ovary cells
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5070371/
https://www.ncbi.nlm.nih.gov/pubmed/27756290
http://dx.doi.org/10.1186/s12896-016-0300-y
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