Exploiting Cell Cycle Phase Specific Expression of Foreign Genes in Perfusion Cultures of Recombinant CHO Cells

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9504840 Kompala This project is on research to characterize the dependence of foreign gene expression on cell cycle phase of recombinant mammalian cells, and to exploit this dependency for maximizing the production of heterologous glycoproteins in high cell density perfusion bioreactors. The three major objectives of the proposed research are: (1) to investigate the hypothesized key role of promoters in determining the cell cycle phase in which the foreign proteins are expressed in recombinant Chinese Hamster Ovary (CHO) cells by using the two-parameter flow cytometic analysis, and to identify and characterize a strong G1 phase-specific promoter for maximizing foreign protein expression at low growth rates; (2) to investigate whether the two opposing trends in the relationship between the specific production rate and the cell growth in continuous suspension cultures of recombinant CHO cultures are caused by G1- and S-phase specific expression from the upstream promoters, and to exploit a strong G1 phase specific promoter to maximize the production of a secreted foreign protein in perfusion bioreactors with attached inclined settlers; and (3) to investigate whether the smaller dying apoptotic cells can be removed before their intracellular glycosidases and proteases are released into the medium. ***