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Abstract Chinese hamster ovary (CHO) cell lines are widely used to manufacture biopharmaceuticals. However, CHO cells are not an optimal expression host due to the intrinsic plasticity of the CHO genome. Genome plasticity can lead to chromosomal rearrangements, transgene exclusion, and phenotypic drift. A poorly understood genomic element of CHO cell line instability is extrachromosomal circular DNA (eccDNA) in gene expression and regulation. EccDNA can facilitate ultra-high gene expression and are found within many eukaryotes including humans, yeast, and plants. EccDNA confers genetic heterogeneity, providing selective advantages to individual cells in response to dynamic environments. In CHO cell cultures, maintaining genetic homogeneity is critical to ensuring consistent productivity and product quality. Understanding eccDNA structure, function, and microevolutionary dynamics under various culture conditions could reveal potential engineering targets for cell line optimization. In this study, eccDNA sequences were investigated at the beginning and end of two-week fed-batch cultures in an ambr ® 250 bioreactor under control and lactate-stressed conditions. This work characterized structure and function of eccDNA in a CHO-K1 clone. Gene annotation identified 1551 unique eccDNA genes including cancer driver genes and genes involved in protein production. Furthermore, RNA-seq data is integrated to identify transcriptionally active eccDNA genes.
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Transcriptomic Insights Into Serum‐Free Medium Adaptation and Temperature Reduction in Chinese Hamster Ovary Cell Cultures
ABSTRACT Chinese hamster ovary (CHO) cells are widely used in recombinant biopharmaceutical production; yet, yields remain low, leading to high market prices. Improving product yield and quality has heavily relied on empirical characterization with limited insight into internal molecular dynamics. RNA‐seq offers a powerful alternative to understand intracellular responses to process changes through gene expression measurement. In this study, three RNA‐seq datasets across three CHO cell lines and four industrially relevant treatments were integrated to characterize the global transcriptome changes, construct a weighted gene co‐expression network, assess the impact on recombinant anti‐interleukin 8 (anti‐IL8) immunoglobulin heavy and light chain transcript abundance, and expression of glycosylation genes. Treatments included adaptation to serum‐free medium, low temperature, low pH, and low glucose concentration in the medium. The findings suggest upregulation of cholesterol biosynthesis is critical for serum‐free medium adaptation, and the rate‐limiting enzymes in the sterol regulatory element‐binding protein pathway (Insig1andSrebf2) could be targeted to accelerate adaptation. Temperature‐induced cell cycle suppression was likely mediated by p53 activation, consistent with previous reports, with the p53‐targets,Zmat3andBtg2, identified as key hub genes. Conversely, glucose and pH were observed to have negligible impacts on the transcriptome. This study uniquely identifies novel genes mediating temperature‐induced cell cycle arrest, distinct glycosylation‐related gene responses impacting product quality, and new stable housekeeping genes for accurate gene expression normalization in CHO cells.
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- PAR ID:
- 10662664
- Publisher / Repository:
- Wiley-VCH
- Date Published:
- Journal Name:
- Biotechnology Journal
- Volume:
- 20
- Issue:
- 7
- ISSN:
- 1860-6768
- Subject(s) / Keyword(s):
- cholesterol metabolism, co-expression analysis, gene expression, RNA-Seq, sterol regulatory element-binding proteins
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1002/biot.70055
