title: Optimization of a quantitative PCR based method for plasmid copy number determination in human cell lines.
abstract: 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.
authors: ACIB, Muthgasse 18, A-1190 Vienna, Austria.
authors: Lukas
authors: L
authors: Fliedl
authors: ACIB, Muthgasse 18, A-1190 Vienna, Austria.
authors: Florian
authors: F
authors: Kast
authors: ACIB, Muthgasse 18, A-1190 Vienna, Austria; Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 18, A-1190 Vienna, Austria.
authors: Johannes
authors: J
authors: Grillari
authors: ACIB, Muthgasse 18, A-1190 Vienna, Austria.
authors: Matthias
authors: M
authors: Wieser
authors: ACIB, Muthgasse 18, A-1190 Vienna, Austria; Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 18, A-1190 Vienna, Austria. Electronic address: regina.grillari@boku.ac.at.
authors: Regina
authors: R
authors: Grillari-Voglauer
mesh_terms: D061249:Batch Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D018628:Gene Dosage / Q000235:genetics*; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D014162:Transfection / Q000379:methods*