Serum-free transfection of CHO cells with chemically defined transfection systems and investigation of their potential for transient and stable transfection
- 177 Downloads
The generation of transgenic cell lines is acquired by facilitating the uptake and integration of DNA. Unfortunately, most of the systems generating stable expression systems are cost and time-consuming and transient expression is optimized to generate milligram amounts of the recombinant protein. Therefore we improved and compared two transfection systems, one based on cationic liposomes consisting of DOTAP/DOPE and the second one on polyethylenimine (PEI). Both systems have been used as chemically defined transfection systems in combination with serum-free cultivated host cell line. At first we had determined the toxicity and ideal ratio of DNA to PEI followed by determination of the optimal transfection conditions in order to achieve maximum transfection efficiency. We then directly compared DOTAP/DOPE and PEI in transient transfection experiments using enhanced green fluorescence protein (EGFP) and a human monoclonal antibody, mAb 2F5, as a model protein. The results which were achieved in case of EGFP were more than 15% transfectants at a viability of 85%. Despite the fact that expression of the mAb was found negligible we used both techniques to generate stable mAb 2F5 expressing cell lines that underwent several cycles of screening and amplification with methotrexate, and resulted in cell lines with similar volumetric production titers. These experiments serve to demonstrate the potential of stable cell lines even in case where the transient systems did not show satisfying results.
KeywordsSerum-free transfection DOTAP/DOPE PEI Antibody expression
This research was part of the Pharma-Planta Project (LSHB-CT-2003-503565), kindly funded by an EU FP6 program.
- Alt FW, Kellems RE, Bertino JR, Schimke RT (1978) Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells. J Biol Chem 253:1357–1370Google Scholar
- Backliwal G, Hildinger M, Chenuet S, DeJesus M, Wurm F (2008) Coexpression of acidic fibroblast growth factor enhances specific productivity and antibody titers in transiently transfected HEK293 cells. Nat Biotechnol 25:162–166Google Scholar
- van der Aa MA, Huth US, Hafele SY, Schubert R, Oosting RS, Mastrobattista E, Hennink WE, Peschka-Suss R, Koning GA, Crommelin DJ (2007) Cellular uptake of cationic polymer-DNA complexes via caveolae plays a pivotal role in gene transfection in COS-7 cells. Pharm Res 24:1590–1598CrossRefGoogle Scholar