An arginase-based system for selection of transfected CHO cells without the use of toxic chemicals
An arginase-based system for selection of transfected CHO cells without the use of toxic chemicals
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DOI：
10.1074/jbc.ra119.011162
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发表时间：
2019-12-06
影响因子：
4.8
通讯作者：
Clynes, Martin
中科院分区：
文献类型：
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作者：
Roca, Berta Capella;Lao, Nga;Clynes, Martin
多胺在细胞增殖、DNA复制、转录和翻译过程中发挥着重要作用，细胞内腐胺、亚精胺和精胺的耗竭会导致细胞生长停滞和最终死亡。CHO-K1细胞的无血清培养基需要补充腐胺，因为这些细胞缺乏多胺生产途径的第一种酶精氨酸酶。在此表型的基础上，我们开发了一个基于精氨酸酶的选择系统。我们用双链载体转染CHO-K1细胞，共表达GFP和精氨酸酶，并在不含l-鸟氨酸和腐胺的培养基中选择细胞，得到稳定表达GFP的混合群体。此外，在这些选择性培养基中，单个克隆稳定表达GFP共42代。利用这种多胺饥饿法，我们获得了重组CHO-K1细胞，该细胞共表达精氨酸酶和人促红细胞生成素（hEPO），也表现出稳定的表达和健康的生长。表达hepo的克隆在商业培养基中生长，如BalanCD和CHO-S无血清培养基（SFM）？II，以及在一个明确的无血清、含腐胺的培养基中培养至少9代（27代），在培养结束时hEPO滴度有最小的下降。我们在几个CHO细胞株（CHO- dp12， CHO- s和DUXB11）和其他哺乳动物细胞系（包括BHK21）中也观察到精氨酸酶活性的缺乏，这表明该选择系统具有更广泛的实用性。总之，我们已经建立了一个易于应用的替代选择系统，可以有效地生成表达生物制药相关蛋白或其他重组蛋白的哺乳动物细胞克隆，而不需要任何有毒的选择剂。我们认为该系统适用于缺乏精氨酸酶活性的哺乳动物细胞系。
Polyamines have essential roles in cell proliferation, DNA replication, transcription, and translation processes, with intracellular depletion of putrescine, spermidine, and spermine resulting in cellular growth arrest and eventual death. Serum-free media for CHO-K1 cells require putrescine supplementation, because these cells lack the first enzyme of the polyamine production pathway, arginase. On the basis of this phenotype, we developed an arginase-based selection system. We transfected CHO-K1 cells with a bicistronic vector co-expressing GFP and arginase and selected cells in media devoid of l-ornithine and putrescine, resulting in mixed populations stably expressing GFP. Moreover, single clones in these selective media stably expressed GFP for a total of 42 generations. Using this polyamine starvation method, we next generated recombinant CHO-K1 cells co-expressing arginase and human erythropoietin (hEPO), which also displayed stable expression and healthy growth. The hEPO-expressing clones grew in commercial media, such as BalanCD and CHO-S serum-free media (SFM)?II, as well as in a defined serum-free, putrescine-containing medium for at least 9 passages (27 generations), with a minimal decrease in hEPO titer by the end of the culture. We observed a lack of arginase activity also in several CHO cell strains (CHO-DP12, CHO-S, and DUXB11) and other mammalian cell lines, including BHK21, suggesting broader utility of this selection system. In conclusion, we have established an easy-to-apply alternative selection system that effectively generates mammalian cell clones expressing biopharmaceutically relevant or other recombinant proteins without the need for any toxic selective agents. We propose that this system is applicable to mammalian cell lines that lack arginase activity.