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Development of Responsive Promoters and their Utilization for Stable CHO Sensor Cell Lines.
Zeh, Nikolas; Otte, Kerstin.
Affiliation
  • Zeh N; Boehringer Ingelheim Pharma GmbH & Co KG, Bioprocess Development Biologicals, Cell Line Development, Biberach, Germany. Nikolas.Zeh@boehringer-ingelheim.com.
  • Otte K; University of Applied Sciences Biberach, Institute of Applied Biotechnology, Biberach, Germany.
Methods Mol Biol ; 2810: 273-284, 2024.
Article de En | MEDLINE | ID: mdl-38926285
ABSTRACT
Chinese hamster ovary (CHO) cells are the most important mammalian expression systems to produce recombinant proteins. To ensure a proper expression of the desired molecule, it is important to monitor and adjust bioprocess parameters like oxygen concentration as well as osmolality. However, the observation of crucial cultivation parameters can be an elaborate procedure requiring lots of hands-on work. In addition, for emerging modeling approaches for bioprocesses, a model cell line responding with a measurable signal to an external influence would be highly valuable. This protocol describes in detail the procedure to generate responsive promoters reacting to limiting conditions as well as the generation of stable sensor cell lines communicating with the operator. Thereby, hypoxia and osmolality sensing response elements established in CHO cells will be utilized to trigger the expression of a minimal CMV promoter. To assess the activity of the responsive promoter in close to real time, unstable variants of GFP and BFP will be expressed, which can be analyzed via flow cytometry. Finally, an automated sampling system coupled to a fluorescence microscope enables a continuous observation of CHO cells and reports emerging limiting conditions by detecting increasing amounts of a specific fluorescent protein.
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Texte intégral: 1 Collection: 01-internacional Base de données: MEDLINE Sujet principal: Cricetulus / Régions promotrices (génétique) Limites: Animals Langue: En Journal: Methods Mol Biol Sujet du journal: BIOLOGIA MOLECULAR Année: 2024 Type de document: Article Pays d'affiliation: Allemagne Pays de publication: États-Unis d'Amérique

Texte intégral: 1 Collection: 01-internacional Base de données: MEDLINE Sujet principal: Cricetulus / Régions promotrices (génétique) Limites: Animals Langue: En Journal: Methods Mol Biol Sujet du journal: BIOLOGIA MOLECULAR Année: 2024 Type de document: Article Pays d'affiliation: Allemagne Pays de publication: États-Unis d'Amérique