Hybrid cell line development system utilizing site-specific integration and methotrexate-mediated gene amplification in Chinese hamster ovary cells
- 주제(키워드) Chinese hamster ovary , CRISPR/Cas9 , DHFR knockout , Gene Amplification , methotrexate , targeted integration
- 주제(DDC) 547
- 발행기관 아주대학교
- 지도교수 이재성
- 발행년도 2023
- 학위수여년월 2023. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000032585
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Site-specific integration has emerged as a promising strategy for streamlined and predictable Chinese hamster ovary (CHO) cell line development (CLD). However, the low specific productivity of the targeted integrants limits their practical applications. In this study, we developed a hybrid CLD platform combining site-specific integration of a transgene and dihydrofolate reductase/methotrexate (DHFR/MTX)-mediated gene amplification to generate high-yield recombinant CHO cell lines. We used the CRISPR/Cas9-based recombinase-mediated cassette exchange landing pad platform to integrate the DHFR expression cassette and transgene landing pad into a CHO genomic hot spot, the C12orf35 locus, of DHFR-knockout CHO-K1 host cell lines. When subjected to various MTX concentrations up to 1 μM, EGFP-expressing targeted integrants showed a 3.6-fold increase in EGFP expression in the presence of 200 nM MTX, accompanied by an increase in DHFR and EGFP copy number. A single-step 200 nM MTX amplification increased the specific monoclonal antibody (mAb) productivity (qmAb) of recombinant mAb-producing targeted integrants by 3-fold, reaching a qmAb of 9.4–12.1 pg/cell/day. Fluorescence in situ hybridization analysis showed colocalization of the DHFR and mAb sequences at the intended chromosomal locations without clear amplified arrays of signals. Most MTX-amplified targeted integrants sustained recombinant mAb production during long-term culture in the absence of MTX, supporting stable gene expression in amplified cell lines. Taken together, these results suggest that the CLD platform can increase the productivity of targeted integrants by amplifying transgene copies.
more목차
1. Introduction 1
2. Materials and Methods 5
2.1. Plasmid construction 5
2.2. Cell lines and culture maintenance 6
2.3. Generation and validation of DHFR− CHO-K1 cell line 6
2.4. Generation of landing pad master cell line 7
2.5. Generation of recombinant mAb-producing rCHO cell lines 8
2.6. MTX amplification 8
2.7. Flow cytometry 8
2.8. Quantitative real-time PCR 9
2.9. Western blot analysis 9
2.10. Fluorescence in situ hybridization analysis 10
2.11. Batch culture 11
2.12. Long-term culture 11
2.13. Statistical analysis 12
3. Results 23
3.1. Generation of DHFR− CHO-K1 cell line 23
3.2. Site-specific integration of DHFR expression cassette and RMCE landing pad 26
3.3. Characterization of DHFR-mediated gene amplification at a defined locus 32
3.4. Characterization of Recombinant mAb-Producing rCHO Cell Pool during DHFR-Mediated Gene Amplification 36
3.5. Evaluation of the efficiency of hybrid cell line development system 43
3.6. Localization of transgenes in recombinant mAb-producing rCHO cells using FISH 45
3.7. Production stability of recombinant mAb-producing rCHO cell pool during long-term cultures in the presence and absence of MTX 48
4. Discussion 51
5. References 57
