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Development of Reporter Systems of Cellular Readouts of Chinese Hamster Ovary (CHO) Cells

Bledowski, Nicole (2022) Development of Reporter Systems of Cellular Readouts of Chinese Hamster Ovary (CHO) Cells. Master of Science by Research (MScRes) thesis, University of Kent,. (doi:10.22024/UniKent/01.02.98518) (KAR id:98518)


Biological stress is perceived and occurs across all cellular organisms. When cells undergo or perceive changes in homeostasis, such as temperature fluctuations, changes in pH/ pressure or oxidative stress, cellular responses are initiated that results in the production of various molecular players to relieve the stress. Different types of stresses cause the specific initiation of diverse molecular pathways and elements to counter the cellular stress. Such stress is perceived in in vitro cultured mammalian cells, such as Chinese hamster ovary (CHO) cells when they are grown in culture to produce secreted biotherapeutic proteins. The perception of different stresses can limit the yield of biotherapeutic protein produced and/or the quality of the protein from such cells. Here, genetic reporter systems that can report on the perception of such stresses have been designed for use in CHO cells during production of biopharmaceuticals so that strategies to prevent or harness such stresses can be developed to enhance recombinant protein production from such cell systems.

A major controlling factor in responding to and combating these cellular stresses begins at the transcriptional level, by the activation of transcription factors (TF), and their complementary DNA binding motifs that regulate gene transcription. The goal of the work presented here was to harness transcription factor binding sites upstream of promoters driving the expression of reporter genes in mammalian cells culture whereby the expression of the fluorescence reporter proteins would provide a real time readout of cellular stress perception that the targeted transcription factor is involved in controlling. Using previously reported transcription factor motif sequences, cloning them into a plasmid vector with selected promoter regions, and multiple cloning sites, reporter gene constructs were developed. A destabilized GFP reporter system was used as the readout as the rapid turnover of this gives a more accurate representation of the current stress perception rather than a historical readout. The reporter constructs were designed, assembled and successfully cloned. The specific stresses and binding sequences utilised targeted transcription factors that were activated upon ER, energy, lipid and oxidative stress. Transfections of a selection of the constructs was then undertaken into CHO-S mammalian cells to establish baseline expression from the reporter constructs as determined by flow cytometry analysis of GFP fluorescence. In particular, carbohydrate response element (ChoRE, energy stress response) and fatty acid stress response (Peroxisome Proliferator-Activated Receptor, PPAR) transcription factor binding sequence containing constructs were transfected and investigated under spent media and oxidative stress conditions. However, under these harsh environmental conditions there was low culture viability and reporter gene expression levels. A further set of transfections were undertaken with the ER stress response (ERSE) TF containing motifs, with varying constructs evaluated that contained different elements, sequence lengths and promotor regions. The cellular external conditions were not altered from the CHO-S maintenance media, and differences in reporter gene expression from these were detected. In conclusion, a toolbox of reporter gene constructs that allow the monitoring in real time of specific stresses on mammalian cells during culture have been designed and developed and some of these have had preliminary validation undertaken. Further analyses can be performed with these under stress conditions to continue to validate and develop a toolbox of stress reporter systems. The potential application of these in CHO cells during the development of cells and bioprocesses for the production of secreted biotherapeutic proteins may help define when such cells perceive different stresses that might limit recombinant protein product yields and quality, allowing the development of systems and processes that can either limit or utilise these responses to further enhance the productivity and quality of product from these cell expression systems.

Item Type: Thesis (Master of Science by Research (MScRes))
Thesis advisor: Smales, Mark
Thesis advisor: Budge, Mark
DOI/Identification number: 10.22024/UniKent/01.02.98518
Uncontrolled keywords: Ampicillin, Base Pair, Carbohydrate Response Element, Cytomegalovirus, Deoxyribonucleic Acid, Endoplasmic Reticulum Stress Element, Fatty Acid Synthase, Gene of Interest, Kanamycin, Kilo Base Pair, Lysogeny Broth, Nuclease-Free Water, Phosphate-Buffered Saline, Polymerase Chain Reaction, Peroxisome Proliferator-Activated Receptor, Ribonucleic Acid, Sodium Dodecyl Sulphate, Sterol Regulatory Element, Simian Virus 40, Tris-Buffered Saline and Tween 20, Transcription Factor
Subjects: Q Science > QR Microbiology
Divisions: Divisions > Division of Natural Sciences > Biosciences
Funders: University of Kent (
SWORD Depositor: System Moodle
Depositing User: System Moodle
Date Deposited: 30 Nov 2022 09:10 UTC
Last Modified: 02 Dec 2022 17:03 UTC
Resource URI: (The current URI for this page, for reference purposes)

University of Kent Author Information

Bledowski, Nicole.

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