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Hybrid cell line development system utilizing site-specific integration and methotrexate-mediated gene amplification in Chinese hamster ovary cells
- Author(s)
- 민홍기
- Alternative Author(s)
- Honggi Min
- Advisor
- 이재성
- Department
- 일반대학원 분자과학기술학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2023-02
- Language
- eng
- Keyword
- CRISPR/Cas9; Chinese hamster ovary; DHFR knockout; Gene Amplification; methotrexate; targeted integration
- Alternative Abstract
- 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.
- Fulltext
- Appears in Collections:
- Graduate School of Ajou University > Department of Molecular Science and Technology > 3. Theses(Master)
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