BAK and BAX deletion using zinc-finger nucleases yields apoptosis-resistant CHO cells
Article first published online: 23 SEP 2009
Copyright © 2009 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 105, Issue 2, pages 330–340, 1 February 2010
How to Cite
Cost, G. J., Freyvert, Y., Vafiadis, A., Santiago, Y., Miller, J. C., Rebar, E., Collingwood, T. N., Snowden, A. and Gregory, P. D. (2010), BAK and BAX deletion using zinc-finger nucleases yields apoptosis-resistant CHO cells. Biotechnol. Bioeng., 105: 330–340. doi: 10.1002/bit.22541
- Issue published online: 21 DEC 2009
- Article first published online: 23 SEP 2009
- Accepted manuscript online: 23 SEP 2009 12:00AM EST
- Manuscript Accepted: 8 SEP 2009
- Manuscript Revised: 10 AUG 2009
- Manuscript Received: 26 JAN 2009
- homologous recombination;
- cell death
Anoxic and metabolic stresses in large-scale cell culture during biopharmaceutical production can induce apoptosis. Strategies designed to ameliorate the problem of apoptosis in cell culture have focused on mRNA knockdown of pro-apoptotic proteins and over-expression of anti-apoptotic ones. Apoptosis in cell culture involves mitochondrial permeabilization by the pro-apoptotic Bak and Bax proteins; activity of either protein is sufficient to permit apoptosis. We demonstrate here the complete and permanent elimination of both the Bak and Bax proteins in combination in Chinese hamster ovary (CHO) cells using zinc-finger nuclease-mediated gene disruption. Zinc-finger nuclease cleavage of BAX and BAK followed by inaccurate DNA repair resulted in knockout of both genes. Cells lacking Bax and Bak grow normally but fail to activate caspases in response to apoptotic stimuli. When grown using scale-down systems under conditions that mimic growth in large-scale bioreactors they are significantly more resistant to apoptosis induced by starvation, staurosporine, and sodium butyrate. When grown under starvation conditions, BAX- and BAK-deleted cells produce two- to fivefold more IgG than wild-type CHO cells. Under normal growth conditions in suspension culture in shake flasks, double-knockout cultures achieve equal or higher cell densities than unmodified wild-type cultures and reach viable cell densities relevant for large-scale industrial protein production. Biotechnol. Bioeng. 2010; 105: 330–340. © 2009 Wiley Periodicals, Inc.