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https://doi.org/10.1186/s12896-016-0300-y
Title: | Evaluating the use of a CpG free promoter for long-term recombinant protein expression stability in Chinese hamster ovary cells | Authors: | Ho, S.C.L Koh, E.Y.C Soo, B.P.C Chao, S.-H Yang, Y |
Keywords: | Acetylation Alkylation Cells Chromosomes Clone cells Cloning Cytology Genes Genetic engineering Mammals Methylation Proteins Recombinant proteins CHO cell DNA Methylation Gene silencing Histone modification Recombinant protein expression Gene expression dinucleotide enhanced green fluorescent protein histone histone deacetylase inhibitor messenger RNA recombinant protein recombinant protein animal cell animal experiment Article cell clone cell culture CHO cell line chromatin immunoprecipitation chromosome controlled study CpG island deacetylation enhancer region female gene dosage gene expression gene interaction gene repression gene silencing genetic analysis genetic linkage histone modification intron mouse nonhuman promoter region protein expression protein stability animal CHO cell line CpG island Cricetulus drug stability evaluation study genetic enhancement genetics isolation and purification metabolism procedures promoter region Animals CHO Cells CpG Islands Cricetulus Drug Stability Genetic Enhancement Promoter Regions, Genetic Recombinant Proteins |
Issue Date: | 2016 | Citation: | Ho, S.C.L, Koh, E.Y.C, Soo, B.P.C, Chao, S.-H, Yang, Y (2016). Evaluating the use of a CpG free promoter for long-term recombinant protein expression stability in Chinese hamster ovary cells. BMC Biotechnology 16 (1) : 71. ScholarBank@NUS Repository. https://doi.org/10.1186/s12896-016-0300-y | Rights: | Attribution 4.0 International | Abstract: | 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. © 2016 The Author(s). | Source Title: | BMC Biotechnology | URI: | https://scholarbank.nus.edu.sg/handle/10635/181334 | ISSN: | 14726750 | DOI: | 10.1186/s12896-016-0300-y | Rights: | Attribution 4.0 International |
Appears in Collections: | Elements Staff Publications |
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