alexa Higher productivity of growth-arrested Chinese hamster ovary cells expressing the cyclin-dependent kinase inhibitor p27.
Bioinformatics & Systems Biology

Bioinformatics & Systems Biology

Journal of Proteomics & Bioinformatics

Author(s): Mazur X, Fussenegger M, Renner WA, Bailey JE

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Abstract We constructed stable Chinese hamster ovary (CHO) cell lines which conditionally and coordinately express the model product gene secreted alkaline phosphatase (SEAP) and one of the cytostatic genes p21, p27, and p53175P, a p53 mutant deficient in apoptotic but not cell-cycle arrest function. The use of dicistronic expression technology allowed the conditional expression of the model product gene and the cytostatic gene in a coordinated fashion from a single expression unit under the control of the tetracycline-responsive promoter PhCMV-1. Due to the presence of a cytostatic gene in the multicistronic expression unit, the growth behavior of the engineered CHO cell lines could be controlled by the addition or withdrawal of the exogenous agent tetracycline to or from the cell culture medium. Withdrawal of tetracycline resulted in sustained growth arrest of the stable cell lines for a prolonged period. The growth arrest of such cell lines was found to be accompanied by a 10-15-fold increase in their production of SEAP per cell. This controlled proliferation technology allows the design of a novel two-stage production process which consists of a proliferation phase leading to the desired cell density, followed by an extended production phase during which the cells remain growth-arrested and increase cell-specific production of a heterologous protein. This article was published in Biotechnol Prog and referenced in Journal of Proteomics & Bioinformatics

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