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Abstract

Using multicistronic expression technology we generated a stable Chinese hamster ovary (CHO) cell line (MG12) expressing a model secreted heterologous glycoprotein, the secreted form of the human placental alkaline phosphatase (SEAP), and bcl-2, best known as an apoptosis inhibitor, in a tetracycline-repressible dicistronic configuration. In batch cultivations in serum-containing medium, MG12 cells reached twice the final viable cell density when Bcl-2 was overexpressed (in the absence oftetracycline) compared to MG12 populations culturedunder tetracycline-containing conditions (bcl-2repressed). However, bcl-2-expressing MG12 cellsshowed no significant retardation of the decline phasecompared to batch cultures in which the dicistronicexpression unit was repressed.Genetic linkage of bcl-2 expression with the reporter protein SEAP in our multicistronic construct allowed online monitoring of Bcl-2 expression over an extended, multistage fixed-bed bioreactor cultivation. The cloned multicistronic expression unit proved to be stable over a 100 day bioreactor run. CHO MG12 cells in the fixed-bed reactor showed a drastic decrease in the release of DNA into the culture supernatant under conditions of reduced tetracycline (and hencederepressed SEAP and bcl-2 overexpression). This observation indicated enhanced robustness associated with bcl-2 overexpression, similar to recent findings for constitutive Bcl-2-overexpressing hybridoma cells under the same bioprocess conditions. These findings indicate, in these serum-containing CHO cell cultures, that overexpression of Bcl-2 results in desirable modifications in culture physiology.

Comparative proteomic analysis of three Chinese hamster ovary (CHO) host cells

“…Over-expression of anti-apoptotic genes, e.g., bcl-2 or bcl-xL , has been tried to increase the viability of CHO cells, thereby the protein production [36, 37]. Meanwhile, gene regulation and culture condition optimization has also been used to induce G1 phase arrest during cell cycle to improve cellular metabolism [38, 39].…”

CHO microRNA engineering is growing up: Recent successes and future challenges

“…Engineering of cell lines has resulted in improved product quality ( Huang et al, 2012a; North et al, 2010; Pouilly et al, 2012; Raju et al, 2001 ) and some improvements in productivity ( Figueroa et al, 2007 ); however, no successes comparable to the enhancements obtained by media optimization have been reported so far, and the most prominent problems of mammalian cell culture, such as the efficient energy utilization ( Zeng et al, 1998 ) have not been completely resolved by metabolic engineering strategies ( Kim and Lee, 2007a,b; Park et al, 2000 ). Most of these metabolic engineering strategies were limited to expressing single genes expected to shift metabolic pathways ( Banmeyer et al, 2004; Hou and Li, 1987a,b; Jeon et al, 2011 ) or to making cells more resistant to apoptosis triggered by nutrient depletion or hyperosmolarity ( Figueroa et al, 2007; Fussenegger et al, 2000; Lim et al, 2006; Park et al, 2000; Sauerwald et al, 2006; Sung et al, 2007; Wong et al, 2006 ). However, cells have redundant mechanisms to control cellular physiology, meaning that cells have different options to reach the same goal ( Charaniya et al, 2009, 2010; Dinnis and James, 2005 ), which has been nicely demonstrated in recent years in several multiparameter -omics studies ( Chong et al, 2010, 2011; Doolan et al, 2010; Meleady et al, 2012b; Wuest et al, 2012; Zhao et al, 2012 ).…”

Evaluating apoptotic gene efficiency for CHO culture performance using targeted integration

“…Conversely, Bcl-2, Bcl-xL and Mcl-1 serve as inhibitor proteins, impeding the generation of these pores (Henry et al, 2020). Many publications report that overexpressing either Bcl-2, Bcl-xL, Mcl-1 or a combination of them, led to higher final titers as apoptosis was significantly delayed (Fussenegger et al, 2000; Majors et al, 2009). Other studies report on knocking out Bax and/or Bak to prevent apoptosis with promising results (Grav et al, 2015; Xiong et al, 2019).…”