Martin Bertschinger

Multiple glycerol shocks increase the calcium phosphate transfection of non-synchronized CHO cells.

The exposure of CHO DG44 cells to an osmotic shock, after DNA uptake, results in a cellular volume decrease of approx. 55%. Repetitive osmotic shocks targeted different sub-populations of cells as was demonstrated using two different fluorescent reporter genes. Also the exposure of a calcium phosphate–DNA coprecipitate to high osmolarity in vitro caused the release of the DNA from the precipitate. The results demonstrate the importance of the osmotic shock on the efficient delivery of plasmid DNA to the nucleus of CHO cells following calcium phosphate-mediated transfection.

Release of Plasmid-DNC from PEI/DNA Particles

The cationic polymer polyethylenimine (PEI) spontaneously forms complexes with DNA that efficiently transfect mammalian cells. To study the reversibility of the DNA/PEI interaction in vitro, preformed complexes were diluted with varying amounts of plasmid DNA and RNA. The released plasmid DNA was visualized by gel electrophoresis. With this simple assay we determined the kinetics of the release of plasmid DNA. Even at very low concentrations RNA instantaneously replaced most of the plasmid DNA in the complex, whereas DNA-mediated release takes up to one day. The results of these in vitro experiments are important for understanding the transport and release of plasmid DNA from PEI/DNA particles after transfection of cells.

The Role of the Sumoylation Pathway in Transient and Stable Recombinant Protein Expression in Chinese Hamster Ovary Cells

One of the essential proteins in the sumoylation pathway is ube2i, one of the subunits of the conjugating complex E2. Transient expression of a short hairpin RNA (shRNA) targeted to the ube2i mRNA in Chinese hamster ovary (CHO) cells increased transient reporter protein expression by 2-3 fold. These results demonstrate the utility of RNAi in studying the role of chromatin modifiers in transgene expression,

Stability and Cytogenetic Characterization of Recombinant CHO Cell Lines Established by Microinjection and Calcium Phosphate Transfection

Chinese hamster ovary cells (CHO) are widely used for the stable production of recombinant proteins. Typically, recombinant cell lines are characterized for the stability of protein expression over a period corresponding to the time needed to scale-up the culture and harvest the product (e.g. 2 to 3 months), for the number of plasmid copies integrated into the host genome, and for the quality and quantity of the recombinant protein. In this study we extended the characterization to the cytogenetic level. Sixteen recombinant CHO cell lines were established using calcium phosphate transfection and microinjection as DNA transfer methods. For each cell line we observed by fluorescence in situ hybridization a single integration site regardless of the gene delivery method, the topology of the DNA (circular or linear), or the integrated plasmid copy number (between 1 and 50). Integration was not targeted to a specific chromosome. Chromosomal rearrangements were observed in about half of these cell lines. This phenomenon occurred independently of the gene transfer method. Interestingly the rearrangements were not on the chromosome where the plasmid integrated. We observed rearrangements between chromosomes and chromosomal imbalances.