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Review Genomic Hotspot Mining and Characterization for Stable Expression of Therapeutic Protein in Chinese Hamster
Xuefeng Ding1, Yazhou Liu1, Lan Luo1
1School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, P. R. China.
Abstract:
The development of rCHO cell lines that stably express therapeutic proteins is crucial for pharmaceutical protein industrial production. In this study, a systematic method was established to identify genomic hotspots for exogenous protein expression in CHO cells and construct stable recombinant CHO cell strains. Four stable monoclonal cell lines (1b7, 1d2, 2d9, and 2f7) were obtained by using the lentiviral random integration reporter gene. Chromosome mapping analysis found four stable integration sites: chr1_0 (7,30,83,299-7,32,45,508 bp) in 1b7, chr1_0 (17,69,68,187-17,69,68,191 bp) in 1d2, chr3 (4,08,81,262-4,08,99,858 bp) in 2d9, and chr5 (1,69,77,575-1,70,61,744 bp) in 2f7. Based on these sites, we developed recombinant CHO cells capable of long-term stable expression of foreign proteins through the combined application of CRISPR/Cas9 technology and Bxb1 recombinase-mediated cassette exchange. Utilizing "promoter capture technology", all screened LP cell monoclonal lines can express exogenous proteins, with the entire construction process completed in just 2∼3 weeks.
Insights
Researchers developed a rapid method to create stable recombinant Chinese hamster ovary (CHO) cell lines for therapeutic protein production. This system identifies genomic hotspots for efficient, long-term expression of foreign proteins.
Area of Science:
- Biotechnology
- Genomics
- Cell Biology
Background:
- Stable recombinant Chinese hamster ovary (CHO) cell lines are essential for industrial pharmaceutical protein production.
- Identifying optimal genomic locations for transgene integration is critical for consistent protein expression.
Purpose of the Study:
- To establish a systematic method for identifying genomic hotspots in CHO cells for exogenous protein expression.
- To construct stable recombinant CHO cell strains utilizing identified hotspots.
Main Methods:
- Lentiviral random integration reporter gene system was used to obtain four stable monoclonal cell lines (1b7, 1d2, 2d9, 2f7).
- Chromosome mapping identified specific integration sites for each cell line.
- CRISPR/Cas9 technology and Bxb1 recombinase-mediated cassette exchange were employed for stable integration.
- Promoter capture technology was utilized for exogenous protein expression.
Main Results:
- Four stable integration sites were identified on chromosomes 1, 3, and 5.
- Recombinant CHO cells capable of long-term stable foreign protein expression were successfully developed.
- The entire construction process, from screening to stable cell line development, was completed within 2-3 weeks.
Conclusions:
- The developed systematic method efficiently identifies genomic hotspots for stable protein expression in CHO cells.
- The combined application of CRISPR/Cas9 and Bxb1 recombinase facilitates rapid construction of high-performance recombinant CHO cell lines.
- This approach significantly accelerates the timeline for producing therapeutic proteins using engineered cell lines.
