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Updated: Jul 6, 2025
The MultiBac Protein Complex Production Platform at the EMBL
Published on: July 11, 2013
Construction and application of a multifunctional CHO cell platform utilizing Cre/lox and Dre/rox site-specific
Chen Zhang1, Feng Chang1, Hui Miao1
1State Key Laboratory of Bioreactor Engineering, School of Biotechnology, East China University of Science and Technology, Shanghai, China.
Abstract:
During the development of traditional Chinese hamster ovary (CHO) cell lines, target genes randomly integrate into the genome upon entering the nucleus, resulting in unpredictable productivity of cell clones. The characterization and screening of high-yielding cell lines is a time-consuming and expensive process. Site-specific integration is recognized as an effective approach for overcoming random integration and improving production stability. We have designed a multifunctional expression cassette, called CDbox, which can be manipulated by the site-specific recombination systems Cre/lox and Dre/rox. The CDbox expression cassette was inserted at the Hipp11(H11) locus hotspot in the CHO-K1 genome using CRISPR/Cas9 technology, and a compliant CHO-CDbox cell platform was screened and obtained. The CHO-CDbox cell platform was transformed into a pool of EGFP-expressing cells using Cre/lox recombinase-mediated cassette exchange (RMCE) in only 2 weeks, and this expression remained stable for at least 75 generations without the need for drug stress. Subsequently, we used the Dre/rox system to directly eliminate the EGFP gene. In addition, two practical applications of the CHO-CDbox cell platform were presented. The first was the quick construction of the Pembrolizumab antibody stable expression strain, while the second was a protocol for the integration of surface-displayed and secreted antibodies on CHO cells. The previous research on site-specific integration of CHO cells has always focused on the single functionality of insertion of target genes. This newly developed CHO cell platform is expected to offer expanded applicability for protein production and gene function studies.
Insights
A new CDbox system enables site-specific gene integration in Chinese hamster ovary (CHO) cells, improving protein production stability and efficiency. This platform accelerates the development of high-yielding cell lines for biopharmaceuticals.
Area of Science:
- Biotechnology
- Molecular Biology
- Cell Line Development
Background:
- Traditional Chinese hamster ovary (CHO) cell line development relies on random gene integration, leading to unpredictable productivity and costly screening.
- Site-specific integration offers a solution for enhanced production stability and consistent yields in CHO cells.
Purpose of the Study:
- To develop a versatile platform for site-specific gene integration in CHO cells.
- To demonstrate the efficiency and stability of the developed platform for protein production.
Main Methods:
- Design and implementation of the multifunctional CDbox expression cassette.
- CRISPR/Cas9 technology for insertion of the CDbox cassette into the CHO-K1 H11 locus.
- Cre/lox and Dre/rox site-specific recombination systems for gene manipulation and exchange.
Main Results:
- Successful generation of a compliant CHO-CDbox cell platform.
- Rapid (2 weeks) and stable expression of EGFP via Cre/lox-mediated cassette exchange, maintained for over 75 generations without drug selection.
- Demonstration of EGFP gene elimination using the Dre/rox system.
- Practical applications shown through rapid construction of a Pembrolizumab antibody stable expression strain and integration of surface-displayed/secreted antibodies.
Conclusions:
- The CHO-CDbox cell platform provides a robust and flexible system for site-specific gene integration in CHO cells.
- This platform significantly improves efficiency and stability in developing high-producing cell lines.
- The developed system offers expanded applicability for biopharmaceutical protein production and gene function studies beyond single-gene insertions.
