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Investigating the role of fatty acids in CHO cell culture and the development of novel genome engineering tools

Avella, Giuseppe (2024) Investigating the role of fatty acids in CHO cell culture and the development of novel genome engineering tools. PhD thesis, Dublin City University.

Abstract
Chinese Hamster ovary (CHO) cells represent one of the most exploited Mammalian cells in the biopharmaceutical industry. They easily proliferate in adherent conditions resembling an epithelium and in suspension conditions. The adaptive process they undergo once depleted of exogenous growth factors and cell-matrix contact is part of this study. We identified the fatty acids (FA) profile of two CHO cell lines, adherent K1 and suspension DG44, as a distribution of saturated, monounsaturated and polyunsaturated FA (SFA/MUFA/PUFA). CHO-DG44 in suspension displayed a lack of PUFA fraction compared to CHO-K1 in adherent conditions, with a heterogeneous profile. Next, we assessed the membrane FA profile remodelling of CHO cells adjusting from adherent to suspension conditions and vice-versa. The results indicated a prompt FAs profile remodelling in both cells over a short timeframe. In addition, the transcriptomic analysis revealed a more detailed overview of the adaptive modifications highlighting metabolic and signalling pathways underlining the differentially expressed genes. Enhancing CHO cell productivity has been an area of intense research to meet the rising market demand. The supplementation of free FA and exogenous lipids in media helps to develop a less hostile environment for the cells, decreasing shear stress, and straightening the membrane. In this work, we genetically manipulated the lipid metabolism of CHO cells by overexpressing key genes involved in lipid pathways. The engineered cells were then supplemented with exogenous lipids and fatty acids, evaluating the impact on productivity of the combination of approaches. Cellular engineering gives more prospects for productivity improvement using genomeengineering tools, allowing the regulation of the activity of proteins before their transcription. Triplex Forming Oligos (TFOs) are proposed in this study as novel genome editing technology, due to their targeting sensibility and flexibility of applications. After testing the natural TFOs targeting eGFP (enhanced Green Fluorescent Protein) plasmid, we evaluated a set of DNA modifications to possibly improve the targeting and stability of the triplex structure.
Metadata
Item Type:Thesis (PhD)
Date of Award:March 2024
Refereed:No
Supervisor(s):Kellett, Andrew and Barron, Niall
Subjects:Biological Sciences > Biochemistry
Humanities > Biological Sciences > Biochemistry
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology
Use License:This item is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 License. View License
Funders:European Union – Horizon 2020 Marie Curie Innovative Training Network (ITN)
ID Code:29420
Deposited On:25 Mar 2024 14:48 by Andrew Kellett . Last Modified 25 Mar 2024 14:48
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Creative Commons: Attribution-Noncommercial-No Derivative Works 4.0
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