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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.

Recombinant CHO cell lines have integrated the expression vectors in various parts of the genome leading to different levels of gene amplification, productivity and stability of protein expression. Identification of insertion sites where gene amplification is possible and the transcription rate is high may lead to systems of site-directed integration and will significantly reduce the process for the generation of stably and highly expressing recombinant cell lines. We have investigated a broad range of recombinant cell lines by FISH analysis and Giemsa-Trypsin banding and analysed their integration loci with regard to the extent of methotrexate pressure, transfection methods, promoters and protein productivities. To summarise, we found that the majority of our high producing recombinant CHO cell lines had integrated the expression construct on a larger chromosome of the genome. Furthermore, except from two cell lines, the exogene was integrated at a single site. The dhfr selection marker was co-localised to the target gene.

We have used a combination of gel electrophoresis and a cell culture assay in microplates to analyse mitogenic activity in tissue extracts. The procedure is a modification of the method described by Kuo et al. The proteins were separated by native gel electrophoresis or isoelectric focusing. The gel was sliced and defined pieces were transferred into tissue culture inserts fitting in 96 well microplates, which contained the test cells. The proteins diffused from the gel slices directly into the culture supernatant and the mitogenic effects were evaluated by a colorimetric assay (MTT or phosphatase activity). Human interleukin 2 was used to demonstrate the feasibility of the method by evaluating the mitogenic effect on the cell line CTLL-2. Extracts of bovine pituitary glands were separated by native gel electrophoresis and isoelectric focusing and several protein bands could be identified which showed a distinct mitogenic effect on human endothelial cells. The method is very sensitive and allows rapid screening of protein mixtures for bioactive fractions.