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可溶性细胞间黏附分子(sICAM)与抗凋亡决定因子bcl-2/bcl-x(L) 的共表达和共扩增对悬浮培养于无血清培养基中的中国仓鼠卵巢细胞DG44(CHO-DG44)的生产力、细胞存活及线粒体数量的影响。

Impact of coexpression and coamplification of sICAM and antiapoptosis determinants bcl-2/bcl-x(L) on productivity, cell survival, and mitochondria number in CHO-DG44 grown in suspension and serum-free media.

作者信息

Meents Heiko, Enenkel Barbara, Eppenberger Hans M, Werner Rolf G, Fussenegger Martin

机构信息

Institute of Biotechnology, Swiss Federal Institute of Technology, ETH Zurich, CH-8093 Zurich, Switzerland.

出版信息

Biotechnol Bioeng. 2002 Dec 20;80(6):706-16. doi: 10.1002/bit.10449.

Abstract

We have engineered dihydrofolate reductase-negative (dhfr-/-) Chinese hamster ovary (CHO) DG44 cells adapted for growth in serum-free suspension cultures for simultaneous expression of the common cold therapeutic, the soluble intercellular adhesion molecule 1 (sICAM), and the antiapoptosis determinants bcl-2 or bcl-x(L). Detailed analyses of titer and antiapoptosis characteristics of these production cell lines included an independent (sICAM; bcl-2/bcl-x(L)) as well as a cocistronic (sICAM-(bcl-2/bcl-x(L))) expression set-up in which translation-initiation of the survival cistron is driven by an internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). In transient transfections or stable mixed populations and in comparison to isogenic sICAM-only control vectors, both bcl-x(L)-encoding configurations achieved higher sICAM yields while bcl-2 over-expression resulted in decreased product levels. Overall, the death-protective impact of bcl-2 and bcl-x(L) in engineered CHO-DG44 was not significant under typical batch-mode operation, an observation that was confirmed by clonal analysis. bcl-2 and bcl-x(L) displayed their antiapoptosis potential only following dhfr-based amplification in sICAM-producing CHO-DG44 cell lines. In all cases, bcl-x(L) outperformed bcl-2 in its cell death-protective capacity. Amplification-dependent high-level expression of mitochondria-localized bcl-2 family members required for successful antiapoptosis engineering may be essential to compensate for increased mitochondria numbers found to be associated with production cell lines grown in serum-free medium.

摘要

我们构建了二氢叶酸还原酶阴性(dhfr-/-)的中国仓鼠卵巢(CHO)DG44细胞,使其适应无血清悬浮培养,用于同时表达普通感冒治疗药物可溶性细胞间粘附分子1(sICAM)和抗凋亡决定因子bcl-2或bcl-x(L)。对这些生产细胞系的滴度和抗凋亡特性进行了详细分析,包括独立(sICAM;bcl-2/bcl-x(L))以及共表达(sICAM-(bcl-2/bcl-x(L)))的表达设置,其中存活顺反子的翻译起始由脑心肌炎病毒(EMCV)的内部核糖体进入位点(IRES)驱动。在瞬时转染或稳定混合群体中,与仅表达sICAM的同基因对照载体相比,两种编码bcl-x(L)的结构均实现了更高的sICAM产量,而bcl-2的过表达导致产物水平降低。总体而言,在典型的分批模式操作下,bcl-2和bcl-x(L)对工程化CHO-DG44的死亡保护作用不显著,这一观察结果通过克隆分析得到证实。bcl-2和bcl-x(L)仅在基于dhfr的扩增后,在产生sICAM的CHO-DG44细胞系中才显示出其抗凋亡潜力。在所有情况下,bcl-x(L)在细胞死亡保护能力方面均优于bcl-2。成功的抗凋亡工程所需的线粒体定位的bcl-2家族成员的扩增依赖性高水平表达,对于补偿与无血清培养基中生长的生产细胞系相关的线粒体数量增加可能至关重要。

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