Application of Endogenous p21 Dependent Transgene Switch in CHO Cell Engineering
Application of Endogenous p21 Dependent Transgene Switch in CHO Cell Engineering
- 주제(키워드) Chinese hamster ovary cells , Endogenous gene tagging , p21 , Transgene expression regulation , Targeted integration , Apoptosis
- 발행기관 아주대학교
- 지도교수 이재성
- 발행년도 2020
- 학위수여년월 2020. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000029993
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Numerous engineering efforts have been made in Chinese hamster ovary (CHO) cells for high level production of therapeutic proteins and overall improvement in cellular properties. However, the dynamic regulation of transgene expression is limited in current systems due to randomly integrated transgenes and limited information on the regulatory elements in CHO cells. In this study, we investigated the effective regulation of transgene expression via targeted integration-based endogenous gene tagging with engineering target genes. Targeted integration of EGFP-human Bcl-2, which encodes an anti-apoptosis effector protein, into the endogenous CHO p21CDKN1A locus effectively reduced the apoptosis in transfected pools of cells and clonal populations, compared with random populations in which human Bcl-2 expression was driven by CMV promoter. Endogenous p21 and EGFP-human Bcl-2 displayed similar expression dynamics in batch cultures, and the anti-apoptotic effect altered the expression pattern of endogenous p21 showing the mutual influences between expression of p21 and Bcl-2. Furthermore, we demonstrated the inducible expression of transgenes by adding low concentrations of hydroxyurea. The present engineering strategy will provide a valuable CHO cell engineering tool that can be used to control dynamic transgene expression in accordance with different cellular states.
more목차
1. Introduction 1
2. Materials and Methods 3
2.1 Cloning vector for TI and random integration 3
2.2 Cell line and cell culture 3
2.3 Genomic DNA extraction and PCR amplification of target regions 4
2.4 Quantitative real time PCR analysis (qRT-PCR) 4
2.5 Chemical treatments 4
2.6 FACS sorting and flow cytometry analysis 5
2.7 Cell cycle analysis 5
2.8 Annexin V staining 5
2.9 Statistical analysis 6
3. Results and discussion 7
3.1 Growth profiles and mRNA expression level of recombinant CHO cell line in batch culture 7
3.2 Vector construction for N/C-Terminus TI and validation of TI 10
3.3 Effect of endogenous tagging with hBcl-2 in condition to induce cell death mediated by apoptosis 12
3.4 Development of cell lines genetically engineered to regulate apoptosis 14
3.5 Characterization of endogenous tagging group and random integration group by analyzing mRNA expression of p21 and transgenes 15
3.6 Development of a p21 inducible condition using Hydroxyurea treatment 18
4. Conclusion 21
5. References 22
SUPPORTING INFORMATION 26
