Serum-free transfection of CHO-cells with tailor-made unilamellar vesicles.
At present, a number of transfection techniques are available to introduce foreign DNA into cells, but still minimal intrusion or interference with normal cell physiology, low toxicity, reproducibility, cost efficiency and successful creation of stable transfectants are highly desirable properties for improved transfection techniques.For all previous transfection experiments done in our labs, using serum-free cultivated host cell lines, an efficiency value of approximately 0.1% for selection of stable cell lines has not been exceeded, consequently we developed and improved a transfection system based on defined liposomes, so-called large unilamellar vesicles, consisting of different lipid compositions to facilitate clone selection and increase the probability for creation of recombinant high-production clones. DNA and DOTAP/DOPE or CHEMS/DOPE interact by electrostatic means forming so-called lipoplexes (Even-Chen and Barenholz 2000) and the lipofection efficiency of those lipoplexes has been determined via confocal microscopy.In addition, the expression of the EGFP was determined by FACS to investigate transient as well as stable transfection and the transfection efficiency of a selection of different commercially available transfection reagents and kits has been compared to our tailor-made liposomes.
Reisinger
Hannes
H
Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences Vienna, Muthgasse 18, 1190, Vienna, Austria, hannes.reisinger@boku.ac.at.
Sevcsik
Eva
E
Vorauer-Uhl
Karola
K
Lohner
Karl
K
Katinger
Hermann
H
Kunert
Renate
R