Effect of different parameters used for in vitro gene electrotransfer on gene expression efficiency, cell viability and visualization of plasmid DNA at the membrane level.

BACKGROUND: Gene electrotransfer is a nonviral method used for DNA delivery into cells. Several steps are involved. One of them is the interaction of DNA with the cell membrane, which is crucial before DNA can enter the cell. We analysed the level of DNA-membrane interaction in relation to electrotransfer efficiency and the importance of the electrophoretic accumulation of DNA at the cell membrane. Systematic comparison of long-duration, short-duration and combinations of electropermeabilizing short (high-voltage; HV) and electrophoretic long (low-voltage; LV) pulses were performed. The effect of Mg(2+) ion concentrations on electrotransfer and their effect on DNase activity were explored. METHODS: To visualize the DNA-membrane interaction, TOTO-1 labeled DNA was used. Transfection efficiency was assessed with plasmid DNA coding for green fluorescent protein. RESULTS: Higher relative electrotransfer efficiency was obtained by using longer pulses, whereas shorter pulses preserved cell viability. Short-duration pulses enabled higher (24%) overall transfection yield compared to long-duration pulses (12%), although a higher DNA-membrane interaction was observed. No significant difference in transfection was obtained between different HV-LV pulsing protocols, although the highest DNA-membrane interaction was observed with HV + LV pulses. The formation of the DNA-membrane complex depended on the Mg(2+) concentration, whereas DNase inhibitor did not affect gene expression. CONCLUSIONS: Gene electrotransfer is a complex phenomenon, where many factors mutually affect the process and the DNA-membrane interaction only comprises the first step. We showed that longer electric pulses are optimal for higher transfection efficiency but reduce viability, whereas shorter pulses enable moderate transfection efficiency and preserve viability. Thus, each application needs a careful choice of pulsing protocol.

Effect of electroporation in a continuous flow system on bioaccumulation of magnesium, zinc and calcium ions in Lactobacillus rhamnosus B 442 cells.

Biomass of Lactobacillus rhamnosus B 442 was subjected to the continuous electroporation using an electroporator with a flow chamber (length of 10 cm, distance between electrodes 0.25 cm, stream width 0.25 cm, flow speed 10 mL/min) to improve accumulation of calcium, magnesium and zinc in the cells. For all tested ions, the following parameters were applied: voltage of 250 V (E = 1 kV/cm), 570 V (E = 2.28 kV/cm), 950 V (E = 3.8 kV/cm), and 1400 V (E = 5.6 kV/cm, the positive control), a frequency of 10 Hz, a pulse width of 100 µs and 30 electrical pulses. The use of PEF increased the accumulation of magnesium, zinc and calcium by 39, 73 and 162%, respectively, compared to the control. Positive correlation was found between ion accumulation and membrane permeability for zinc and magnesium. For calcium, the initial increase in permeability resulted in higher ion accumulation, but with a further increase of this parameter at 3.8 kV/cm, its decrease was observed caused by a drop in cell viability. Total number of bacteria ranged from 1.67 × 108 (for the cultures supplemented with calcium) to 1.34 × 1012 cfu/mL (for the cultures supplemented with magnesium).

Evaluation and Optimization of Protein Extraction From E. coli by Electroporation.

Growing diversity of protein-based technologies dictates further development of bio manufacturing to lower the cost of production and maximize yields. Intracellularly expressed recombinant proteins must be extracted from production host prior to purification. Use of electroporation to obtain proteins from bacteria and yeasts has been demonstrated in several studies for different modes of operation and formats. Here we tested various protocols for protein extraction from Escherichia coli by means of electroporation. The tested protocols were compared to established extraction methods of ultrasonication and glass-bead milling in terms of protein yields and content of impurities such as host cell DNA and endotoxins in the lysate. Protein extraction yield was maximal when exponentially growing bacteria were treated at 37°C, regardless of the electroporation mode of operation (batch or flow). We were unable to eliminate co-extraction of host DNA and endotoxins, but with 8 × 1 ms, 5 kV/cm, 1 Hz pulses they were minimized. Yields with optimized electroporation (up to 86 g protein/kg dry weight) were inferior to those in ultrasonication (up to 144 g protein/kg dry weight) and glass-bead milling (up to 280 g protein/kg dry weight). Nevertheless, electroporation largely avoids cell lysis and disintegration with which the extract is a mix of extracted proteins with debris of the bacterial envelope and bacterial DNA, which necessitates further purification.

Electroporation as a Solvent-Free Green Technique for Non-Destructive Extraction of Proteins and Lipids From Chlorella vulgaris.

Proteins extracted from microalgae for food, personal care products and cosmetics must be of high purity, requiring solvent-free extraction techniques despite their generally considerably lower protein yield and higher energy consumption. Here, three such approaches for green extraction of proteins from Chlorella vulgaris were evaluated: ultrasound, freeze-thawing, and electroporation; chemical lysis was used as positive control (maximal achievable extraction), and no extraction treatment as negative control. Compared to chemical lysis, electroporation yielded the highest fraction of extracted protein mass in the supernatant (≤27%), ultrasound ≤24%, and freeze-thawing ≤15%. After a growth lag of several days, electroporated groups of algal cells started to exhibit growth dynamics similar to the negative control group, while no growth regeneration was detected in groups exposed to ultrasound, freeze-thawing, or chemical lysis. For electroporation as the most efficient and the only non-destructive among the considered solvent-free protein extraction techniques, simultaneous extraction of intracellular algal lipids into supernatant was then investigated by HPLC, proving relatively low-yield (≤7% of the total algal lipid mass), yet feasible for glycerides (tri-, di-, and mono-) as well as other fatty acid derivatives. Our results show that electroporation, though lower in extraction yields than chemical lysis or mechanical disintegration, is in contrast to them a technique for largely debris-free extraction of proteins from microalgae, with no need for prior concentration or drying, with feasible growth regeneration, and with potential for simultaneous extraction of intracellular algal lipids into the supernatant.

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.

Techniques of signal generation required for electropermeabilization. Survey of electropermeabilization devices.

Electropermeabilization is a phenomenon that transiently increases permeability of the cell plasma membrane. In the state of high permeability, the plasma membrane allows ions, small and large molecules to be introduced into the cytoplasm, although the cell plasma membrane represents a considerable barrier for them in its normal state. Besides introduction of various substances to cell cytoplasm, permeabilized cell membrane allows cell fusion or insertion of proteins to the cell membrane. Efficiency of all these applications strongly depends on parameters of electric pulses that are delivered to the treated object using specially developed electrodes and electronic devices--electroporators. In this paper we present and compare most commonly used techniques of signal generation required for electropermeabilization. In addition, we present an overview of commercially available electroporators and electroporation systems that were described in accessible literature.

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.

Electrofusion of B16-F1 and CHO cells: the comparison of the pulse first and contact first protocols.

High voltage electric pulses induce permeabilisation (i.e. electroporation) of cell membranes. Electric pulses also induce fusion of cells which are in contact. Contacts between cells can be established before electroporation, in so-called contact first or after electroporation in pulse first protocol. The lowest fusion yield was obtained by pulse first protocol (0.8%±0.3%) and it was only detected by phase contrast microscopy. Higher fusion yield detected by fluorescence microscopy was obtained by contact first protocol. The highest fusion yield (15%) was obtained by modified adherence method whereas fusion yield obtained by dielectrophoresis was lower (4%). The results are in agreement with current understanding of electrofusion process and with existing electrochemical models. Our data indicate that probability of stalk formation leading to fusion pores and cytoplasmic mixing is higher in contact first protocol where cells in contact are exposed to electric pulses. Another contribution of present study is the comparison of two detection methods. Although fusion yield can be more precisely determined with fluorescence microscopy we should note that by using this detection method single coloured fused cells cannot be detected. Therefore low fusion yields are more reliably detected by phase contrast microscopy.

Metastatic potential of melanoma cells is not affected by electrochemotherapy.

Electrochemotherapy is a local treatment combining chemotherapy and application of electric pulses to the tumour. Electrochemotherapy with bleomycin and cisplatin has shown its effectiveness in controlling local tumour growth in the treatment of malignant melanoma. However, the effect of electrochemotherapy on the metastatic potential of tumour cells is not known. Prevention of metastasis is an important aspect of successful treatment; however, it is known that metastasis can be induced by different treatment modalities. Therefore, the aim of this study was to evaluate the effect of electrochemotherapy with cisplatin on the metastatic potential of human malignant melanoma cells. Cells treated by electrochemotherapy with cisplatin were tested for their ability to migrate and invade through Matrigel-coated porous membrane. In addition, RNA was isolated from cells after treatment and differentially expressed genes were investigated by microarray analysis to evaluate the effect of electrochemotherapy with cisplatin on gene expression. There were no significant changes observed in cell migration and invasion of melanoma cells after electrochemotherapy. In addition, there were no changes observed in cell adhesion on Matrigel. Gene expression analysis showed that a very low number of genes were differentially expressed after electrochemotherapy with cisplatin. Two genes, LAMB3 and CD63 involved in cell migration, were both downregulated after electrochemotherapy with cisplatin and the expression of metastasis promoting genes was not increased after electrochemotherapy. Our data suggest that electrochemotherapy does not increase the metastatic behaviour of human melanoma cells.