Browsing by Autor "Eric Kitas"

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Synergistic Enhancement of Transient Expression by Dioleoyl-Melittin (DOM) and Polyethylenimine (PEI) in Mammalian Cells in Suspensionculture
(Springer Science+Business Media, 2006) E.-J. Schlaeǵer; Jean‐Yves Legendre; Anna M. Trzeciak; Eric Kitas; Klaus Christensen; Ulrich Deuschle; A. Supersaxo
Dioleoyl-melittin, which is a conjugate of dioleoyl-phosphatidyl-ethanolamine-N-3-(2-pyridyldithio) propionate with the amphipatic peptide melittin Gly-Cys 1 represents a new class of peptide-based reagent for efficient transfection of mammalian cells. In this work we investigated the transfection efficiency of dioleoyl-melittin (DOM) combined with polyethylenimine (PEI) using HEK293(EBNA) cells grown in serum-free suspension cultures. Gene expression was monitored using the luciferase reporter gene and the human soluble TNF receptor p55 gene (TNFRp55) inserted into different vectors. Our data clearly show that DOM together with PEI exhibited synergistic enhancement for gene expression in EBNA cells. At the optimal DNA-DOM-PEI weight ratio the efficiency of transfection increases significantly compared to corresponding PEI and DOM transfection at low DNA concentration. In summary, our data show that dioleoyl-melittin and polyethylenimine act synergistically in transfecting 293(EBNA) cells grown in serum-free suspension culture.
Transient Transfection in Mammalian Cells
(Springer Science+Business Media, 2006) E.-J. Schlaeǵer; Jean‐Yves Legendre; Anna M. Trzeciak; Eric Kitas; Klaus Christensen; Ulrich Deuschle; A. Supersaxo
The aim of the presented work was to develop a cost-effective and easily scaleable transient transfection system with mammalian cells grown in serum-free suspension culture. For this purpose the cationic polyethylenimine (PEI) and the novel hybrid molecule dioleoyl-melittin (DOM) were used. Both substances are highly efficient transfection reagents for mammalian cells and have been described recently. The transfection complexes were made directly within the cell culture by consecutively adding plasmid and PEI (direct method). Alternatively, the DNA-PEI transfection complexes were performed in fresh medium (1/10 culture volume) and then added to the cells (indirect method). Here we have optimized the PEI-mediated transfection conditions for HEK293 and 293EBNA cells grown in serum-free suspension culture for large scale experiments. In a second part of the investigation the efficiency of gene expression was studied in labscale as well as in fermentor scale by combining DOM with PEI to form transfection complexes. Our data show the PEI-mediated transfection into EBNA cells can be used as a cost-effective transfection system in spinner culture scale up to 2-5L, to produce milligram amounts of recombinant protein. Fermentor scale experiments, however, are less efficient because the PEI- mediated transient gene expression is inhibited by the conditioned medium. Transfection experiments performed in the present of both DOM and PEI exhibited synergistic enhancement of gene expression in EBNA cells. The synergistic enhancement between DOM and PEI at low dose DNA (0, l-0,2μg/ml) was seen in spinner scale as well as in 10L and 23L fermentor