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Endogenous BiP reporter system for simultaneous identification of ER stress and antibody production in Chinese hamster ovary cells
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 이재성 | - |
| dc.contributor.author | 경민지 | - |
| dc.date.accessioned | 2022-11-29T03:01:30Z | - |
| dc.date.available | 2022-11-29T03:01:30Z | - |
| dc.date.issued | 2022-02 | - |
| dc.identifier.other | 31518 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/21240 | - |
| dc.description | 학위논문(석사)--아주대학교 일반대학원 :분자과학기술학과,2022. 2 | - |
| dc.description.tableofcontents | 1. Introduction 1 2. Materials and Methods 4 2.1. Cloning of sgRNA/Cas9 vector and donor plasmid 4 2.2. Cell line development (CLD) 5 2.3. Cell culture maintenance 6 2.4. Chemical treatment 6 2.5. Cell imaging 7 2.6. Batch culture 7 2.7. Analysis of mGFP expression level using flow cytometry 7 2.8. Western blot analysis 8 2.9. Measurement of mAb concentration by ELISA 9 2.10. Preparation of genomic DNA, RNA, and cDNA 9 2.11. Evaluation of the relative HC, LC gene copies, and mRNA expression level using quantitative real-time polymerase chain reaction (qRT-PCR) 10 2.12. Statistical analysis 10 3. Results 17 3.1. CRISPR/Cas9-mediated targeted integration of mGFP into the endogenous HSPA5 (BiP) locus generated an ER stress monitoring system in CHO cells 17 3.2. BiP-mGFP cell line exhibited functional UPR activation that was directly measured using fluorescence levels 23 3.3. Basal expression levels of BiP affect recombinant protein productivity during random integration-based CLD 28 3.4. Basal expression levels of BiP affect recombinant protein productivity during site-specific integration-based CLD 31 3.5. Basal expression levels of BiP implied potential differences in production capacity in clonally derived rCHO cell line 41 4. Discussion 45 5. References 50 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | Endogenous BiP reporter system for simultaneous identification of ER stress and antibody production in Chinese hamster ovary cells | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.department | 일반대학원 분자과학기술학과 | - |
| dc.date.awarded | 2022. 2 | - |
| dc.description.degree | Master | - |
| dc.identifier.localId | 1245068 | - |
| dc.identifier.uci | I804:41038-000000031518 | - |
| dc.identifier.url | https://dcoll.ajou.ac.kr/dcollection/common/orgView/000000031518 | - |
| dc.subject.keyword | BiP | - |
| dc.subject.keyword | Cell engineering | - |
| dc.subject.keyword | ER stress | - |
| dc.subject.keyword | Recombinant protein production | - |
| dc.subject.keyword | Unfolded protein response | - |
| dc.description.alternativeAbstract | As the biopharmaceutical industry expands, improving the production of therapeutic proteins using Chinese hamster ovary (CHO) cells is important. However, excessive and complicated protein production causes protein misfolding and triggers endoplasmic reticulum (ER) stress. When ER stress occurs, cells mediate the unfolded protein response (UPR) pathway to restore protein homeostasis and folding capacity of the ER. However, when the cells fail to control prolonged ER stress, UPR induces apoptosis. Therefore, monitoring the degree of UPR is required to achieve high productivity and the desired quality. In this study, we developed a fluorescence-based UPR monitoring system for CHO cells. We integrated mGFP into endogenous HSPA5 encoding BiP, a major ER chaperone, and the primary ER stress activation sensor, using CRISPR/Cas9-mediated targeted integration. The mGFP expression level changed according to the ER stress induced by chemical treatment and batch culture in the engineered cell line. Using this monitoring system, we demonstrated that host cells and recombinant CHO cell lines with different mean fluorescent intensities (MFI; basal expression levels of BiP) possess a distinct capacity for stress culture conditions induced by recombinant protein production. Antibody-producing recombinant CHO cell lines were generated using site-specific integration based on host cells equipped with the BiP reporter system. Targeted integrants showed a strong correlation between productivity and MFI, reflecting the potential of this monitoring system as a screening readout for high producers. Taken together, these data demonstrate the utility of the endogenous BiP reporter system for the detection of real-time dynamic changes in endogenous UPR and its potential for applications in recombinant protein production during CHO cell line development. | - |
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- Graduate School of Ajou University > Department of Molecular Science and Technology > 3. Theses(Master)
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