BAK and BAX Deletion Using Zinc-Finger Nucleases Yields Apoptosis-Resistant CHO Cells
BAK and BAX Deletion Using Zinc-Finger Nucleases Yields Apoptosis-Resistant CHO Cells
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DOI：
10.1002/bit.22541
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发表时间：
2010-02-01
影响因子：
3.8
通讯作者：
Gregory, Philip D.
中科院分区：
文献类型：
--
作者：
Cost, Gregory J.;Freyvert, Yevgeniy;Gregory, Philip D.
生物制药生产过程中大规模细胞培养中的缺氧和代谢应激可诱导细胞凋亡。旨在改善细胞培养中细胞凋亡问题的策略集中在促凋亡蛋白的mRNA敲低和抗凋亡蛋白的过度表达上。细胞培养物中的细胞凋亡涉及促细胞凋亡巴克和Bax蛋白的线粒体透化;任一蛋白的活性足以允许细胞凋亡。我们在这里证明了在中国仓鼠卵巢（CHO）细胞中使用锌指核酸酶介导的基因破坏完全和永久消除巴克和Bax蛋白的组合。BAX和巴克的锌指核酸酶切割，随后是不准确的DNA修复，导致两个基因的敲除。缺乏Bax和巴克的细胞正常生长，但无法激活半胱天冬酶以响应细胞凋亡刺激。当在模拟大规模生物反应器中生长的条件下使用按比例缩小的系统生长时，它们对由饥饿、星形孢菌素和丁酸钠诱导的细胞凋亡具有显著更强的抗性。当在饥饿条件下生长时，BAX和BAK缺失的细胞产生比野生型CHO细胞多2 - 5倍的IgG。在摇瓶中悬浮培养的正常生长条件下，双敲除培养物达到与未修饰的野生型培养物相等或更高的细胞密度，并达到与大规模工业蛋白质生产相关的活细胞密度。Biotechnol. Bioeng. 2010;105：330-340. (C)2009 Wiley Periodicals，Inc.
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 wildtype 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. (C) 2009 Wiley Periodicals, Inc.