Fussenegger M, Schlatter S, Dätwyler D, Mazur X, Bailey J E
Institute of Biotechnology, Swiss Federal Institute of Technology, Zurich, Switzerland.
Nat Biotechnol. 1998 May;16(5):468-72. doi: 10.1038/nbt0598-468.
The eukaryotic cell cycle is regulated by a complex network of many proteins. Effective reprogramming of this complex regulatory apparatus to achieve bioprocess goals, such as cessation of proliferation at high cell density to allow an extended period of high production, can require coordinated manipulation of multiple genes. Previous efforts to establish inducible cell-cycle arrest of Chinese hamster ovary (CHO) cells by regulated expression of the cyclin-dependent kinase inhibitor (CDI) p21 failed. By tetracycline-regulated coexpression of p21 and the differentiation factor CCAAT/enhancer-binding protein alpha (which both stabilizes and induces p21), we have achieved effective cell-cycle arrest. Production of a model heterologous protein (secreted alkaline phosphatase; SEAP) has been increased 10-15 times, on a per cell basis, relative to an isogenic control cell line. Because activation of apoptosis response is a possible complication in a proliferation-arrested culture, the survival gene bcl-xL was coexpressed with another CDI, p27, found to enable CHO cell-cycle arrest predominantly in G1 phase. CHO cells stably transfected with a tricistronic construct containing the genes for these proteins and for SEAP showed 30-fold higher SEAP expression than controls.
真核细胞周期受多种蛋白质组成的复杂网络调控。要有效重编程这一复杂调控机制以实现生物工艺目标，比如在高细胞密度时停止增殖以延长高产期，可能需要对多个基因进行协同操控。此前通过调控表达细胞周期蛋白依赖性激酶抑制剂（CDI）p21来诱导中国仓鼠卵巢（CHO）细胞发生细胞周期阻滞的尝试失败了。通过四环素调控的p21与分化因子CCAAT/增强子结合蛋白α（既能稳定p21又能诱导其表达）的共表达，我们实现了有效的细胞周期阻滞。与同基因对照细胞系相比，在每个细胞基础上，一种模型异源蛋白（分泌型碱性磷酸酶；SEAP）的产量提高了10至15倍。由于在增殖阻滞的培养物中激活凋亡反应可能是一个并发症，存活基因bcl-xL与另一种CDI p27共表达，发现p27能使CHO细胞周期主要阻滞在G1期。用包含这些蛋白质及SEAP基因的三顺反子构建体稳定转染的CHO细胞，其SEAP表达比对照高30倍。
