Pulsed Electric Fields Alter Expression of NF-κB Promoter-Controlled Gene.

The possibility to artificially adjust and fine-tune gene expression is one of the key milestones in bioengineering, synthetic biology, and advanced medicine. Since the effects of proteins or other transgene products depend on the dosage, controlled gene expression is required for any applications, where even slight fluctuations of the transgene product impact its function or other critical cell parameters. In this context, physical techniques demonstrate optimistic perspectives, and pulsed electric field technology is a potential candidate for a noninvasive, biophysical gene regulator, exploiting an easily adjustable pulse generating device. We exposed mammalian cells, transfected with a NF-κB pathway-controlled transcription system, to a range of microsecond-duration pulsed electric field parameters. To prevent toxicity, we used protocols that would generate relatively mild physical stimulation. The present study, for the first time, proves the principle that microsecond-duration pulsed electric fields can alter single-gene expression in plasmid context in mammalian cells without significant damage to cell integrity or viability. Gene expression might be upregulated or downregulated depending on the cell line and parameters applied. This noninvasive, ligand-, cofactor-, nanoparticle-free approach enables easily controlled direct electrostimulation of the construct carrying the gene of interest; the discovery may contribute towards the path of simplification of the complexity of physical systems in gene regulation and create further synergies between electronics, synthetic biology, and medicine.

Detection of intracellular biomarkers in viable cells using millisecond pulsed electric fields.

Reversible electroporation is a temporary permeabilization of cell membrane through the formation of transient pores created by short high voltage electric pulses. This method has numerous applications in biology and biotechnology and has become an important technique in molecular medicine. Reversible electroporation is usually used to transfer macromolecules into the cells. However, the delivery of large molecules such as proteins into cells without loss of cell viability remains a challenge. In our study, we investigated whether electroporation can be used for this purpose. The study was performed with the primary mouse splenocytes and Jurkat cell line. The electroporation efficacy was evaluated by flow cytometry. We used the reversible electroporation for intracellular marker detection investigating antibody and fluorescein-conjugated dextran transfer efficiency, cell viability and metabolic activity. We have found that reversible electroporation parameters can be optimized for efficient transfer of large molecules such as antibodies/proteins into live cells without a significant loss of cell viability. We conclude that a well-established and relatively easy method of reversible electroporation can be adjusted to detect intracellular biomarkers in viable cells. This is a new approach on how electroporation could be utilised in medicine and biological research to detect rare subpopulations of cells that produce specific markers and to keep cells viable. This would allow the use of these rare subpopulations of isolated cells for further research and personalized medicine.