Untersuchungen zum nichtviralen Gentransfer mittels integrierender und extrachromosomal-replikationskompetenter Plasmid-DNA in hämatopoetische Zellen

Abstract

The overall aim of this thesis was the optimization of nonviral gene transfer particularly into hematopoietic cells. High transfection efficiencies and transgene expressions were achieved by the use of plasmid DNA (pDNA) concatemers. In further parts of the study it was investigated to which extend stable transgene expression could be achieved either with persistent extrachromosomal pDNA or with integrated pDNA into the genome of hematopoietic cells. The investigation of a pDNA dimer in comparison to its monomer resulted in significant differences regarding intracellular localization and transgene expression after transfection. When equimolar ratios of gene copies were transfected, no difference was found with respect to EGFP expression per transfected cell. Interestingly higher EGFP expression was found in dimer-transfected cells, when equimolar ratios of plasmid molecules were transfected. Further, the intracellular localization of the different constructs was investigated after transfection of equimolar ratios of either gene copies or pDNA molecules. From the relative amounts of pDNA present in the nucleus and the resulting transgene expression the relative transcription efficiency was calculated. Interestingly the dimeric pEGFP-N1 molecule mediated transgene expression with a 3.5-fold higher efficiency compared to its monomer.