The impact of impaired DNA mobility on gene electrotransfer efficiency: analysis in 3D model.

BACKGROUND: Gene electrotransfer is an established method that enables transfer of DNA into cells with electric pulses. Several studies analyzed and optimized different parameters of gene electrotransfer, however, one of main obstacles toward efficient electrotransfection in vivo is relatively poor DNA mobility in tissues. Our aim was to analyze the effect of impaired mobility on gene electrotransfer efficiency experimentally and theoretically. We applied electric pulses with different durations on plated cells, cells grown on collagen layer and cells embedded in collagen gel (3D model) and analyzed gene electrotransfer efficiency. In order to analyze the effect of impaired mobility on gene electrotransfer efficiency, we applied electric pulses with different durations on plated cells, cells grown on collagen layer and cells embedded in collagen gel (3D model) and analyzed gene electrotransfer efficiency. RESULTS: We obtained the highest transfection in plated cells, while transfection efficiency of embedded cells in 3D model was lowest, similarly as in in vivo. To further analyze DNA diffusion in 3D model, we applied DNA on top or injected it into 3D model and showed, that for the former gene electrotransfer efficiency was similarly as in in vivo. The experimental results are explained with theoretical analysis of DNA diffusion and electromobility. CONCLUSION: We show, empirically and theoretically that DNA has impaired electromobility and especially diffusion in collagen environment, where the latter crucially limits electrotransfection. Our model enables optimization of gene electrotransfer in in vitro conditions.

Introduction of Phage Genome into Escherichia coli by Electroporation.

Electroporation has been an established tool for DNA delivery into prokaryotic and eukaryotic cells, thus facilitating basic research studies and improving medical treatments. Here we describe its use for introduction of phage genomic DNA into Escherichia coli cells, including preparation of electrocompetent cells, electric pulse optimization and recovery of electrotransformed cells. The technique can also be adapted for other bacterial species.

Gene Electrotransfer: A Mechanistic Perspective.

Gene electrotransfer is a powerful method of DNA delivery offering several medical applications, among the most promising of which are DNA vaccination and gene therapy for cancer treatment. Electroporation entails the application of electric fields to cells which then experience a local and transient change of membrane permeability. Although gene electrotransfer has been extensively studied in in vitro and in vivo environments, the mechanisms by which DNA enters and navigates through cells are not fully understood. Here we present a comprehensive review of the body of knowledge concerning gene electrotransfer that has been accumulated over the last three decades. For that purpose, after briefly reviewing the medical applications that gene electrotransfer can provide, we outline membrane electropermeabilization, a key process for the delivery of DNA and smaller molecules. Since gene electrotransfer is a multipart process, we proceed our review in describing step by step our current understanding, with particular emphasis on DNA internalization and intracellular trafficking. Finally, we turn our attention to in vivo testing and methodology for gene electrotransfer.

Testing a prototype pulse generator for a continuous flow system and its use for E. coli inactivation and microalgae lipid extraction.

Among other applications, electroporation is used for the inactivation of pathogens and extraction of substances from microorganisms in liquids where large scale flow systems are used. The aim of our work was therefore to test a pulse generator that enables continuous pulsed electric field (PEF) treatment for Escherichia coli inactivation and microalgae lipid extraction. In the continuous flow PEF system, the flow rate was adjusted so that each bacterial cell received a defined number of pulses. The results of PEF flow treatment showed that the number of pulses influences E. coli inactivation to the same extent as in the previously described cuvette system, i.e., batch system. The continuous flow PEF system was also tested and evaluated for lipid extraction from microalgae Chlorella vulgaris. In control experiments, lipids were extracted via concentration of biomass, drying and cell rupture using pressure or an organic solvent. In contrast, electroporation bypasses all stages, since cells were directly ruptured in the broth and the oil that floated on the broth was skimmed off. The initial experiments showed a 50% oil yield using the electroporation flow system in comparison to extraction with organic solvent.