中国仓鼠卵巢细胞
转染
聚乙烯亚胺
分子生物学
重组DNA
幼仓鼠肾细胞
细胞培养
化学
DNA
生物
生物化学
细胞
基因
遗传学
作者
Madiha Derouazi,Philippe Girard,Frédéric Van Tilborgh,Keyvan Iglesias,Natalie Muller,Martin Bertschinger,Florian M. Wurm
摘要
To date, methods for large-scale transient gene expression (TGE) in cultivated mammalian cells have focused on two transfection vehicles: polyethylenimine (PEI) and calcium phosphate (CaPi). Both have been shown to result in high transfection efficiencies at scales beyond 10 L. Unfortunately, both approaches yield higher levels of recombinant protein (r-protein) in the presence of serum than in its absence. Since serum is a major cost factor and an obstacle to protein purification, our goal was to develop a large-scale TGE process for Chinese hamster ovary (CHO) cells in the absence of serum. CHO-DG44 cells were cultivated and transfected in a chemically defined medium using linear 25 kDa PEI as a transfection vehicle. Parameters that were optimized included the DNA amount, the DNA-to-PEI ratio, the timing and solution conditions for complex formation, the transfection medium, and the cell density at the time of transfection. The highest levels of r-protein expression were observed when cultures at a density of 2.0 × 106 cells/ml were transfected with 2.5 μg/ml DNA in RPMI 1640 medium containing 25 mM HEPES at pH 7.1. The transfection complex was formed at a DNA:PEI ratio of 1:2 (w/w) in 150 mM NaCl with a 10-min incubation at room temperature prior to addition to the culture. The procedure was scaled up for a 20-L bioreactor, yielding expression levels of 10 mg/l for an intracellular protein and 8 mg/l for a secreted antibody. © 2004 Wiley Periodicals, Inc.
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