Fliedl Lukas, Kast Florian, Grillari Johannes, Wieser Matthias, Grillari-Voglauer Regina
ACIB, Muthgasse 18, A-1190 Vienna, Austria.
ACIB, Muthgasse 18, A-1190 Vienna, Austria; Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 18, A-1190 Vienna, Austria.
N Biotechnol. 2015 Dec 25;32(6):716-9. doi: 10.1016/j.nbt.2015.03.004. Epub 2015 Mar 18.
Transient gene expression (TGE) is an essential tool for the production of recombinant proteins, especially in early drug discovery and development phases of biopharmaceuticals. The need for fast production of sufficient recombinant protein for initial tests has dramatically increased with increase in the identification of potential novel pharmaceutical targets. One of the critical factors for transient transfection is plasmid copy number (PCN), for which we here provide an optimized qPCR based protocol. Thereby, we show the loss of PCN during a typical batch process of HEK293 cells after transfection from 606,000 to 4560 copies per cell within 5 days. Finally two novel human kidney cell lines, RS and RPTEC/TERT1 were compared to HEK293 and proved competitive in terms of PCN and specific productivity. In conclusion, since trafficking and degradation of plasmid DNA is not fully understood yet, improved methods for analysis of PCN may contribute to design specific and more stable plasmids for high yield transient gene expression systems.
瞬时基因表达（TGE）是生产重组蛋白的重要工具，尤其在生物制药的早期药物发现和开发阶段。随着潜在新型药物靶点识别数量的增加，为初始测试快速生产足够重组蛋白的需求急剧上升。瞬时转染的关键因素之一是质粒拷贝数（PCN），我们在此提供一种基于qPCR的优化方案。由此，我们展示了在HEK293细胞典型的分批培养过程中，转染后5天内每个细胞的质粒拷贝数从606,000降至4560。最后，将两种新型人肾细胞系RS和RPTEC/TERT1与HEK293进行比较，结果表明它们在质粒拷贝数和比生产率方面具有竞争力。总之，由于质粒DNA的运输和降解尚未完全明确，改进的质粒拷贝数分析方法可能有助于设计用于高产瞬时基因表达系统的特定且更稳定的质粒。
