DNA transfection of Escherichia coli by electroporation

Electroporation was applied to transfection and transformation of Escherichia coli. Efficient transfer of DNA was achieved by a single voltage pulse at 2.5 kV (initial electric field strength = 6.25 kV/cm), with a 25 microF capacitor. As the recipient for transfecting DNA in the electroporation, spheroplasts, EDTA-treated cells and osmotically shocked bacteria were inferior to intact E. coli. Various parameters affecting the transfection efficiency were defined including growth phase of recipient cells, concentrations of DNA and cells, temperature and additions. In most strains tested, electroporation was far more efficient than Ca2+-dependent transfection (transformation). Various aspects of the electroporation-mediated DNA uptake are discussed.     

High-efficiency gene transfection by in situ electroporation of cultured cells

It is demonstrated in this study that high-efficiency gene transfection can be obtained by directly electroporating cultured mammalian cells in their attached state using a pulsed radio-frequency (RF) electric field. A plasmid DNA containing the reporter gene beta-gal was introduced into COS-M6 cells and CV-1 cells using this in situ electroporation method. At the optimal electric field strength (1.2 kV/cm), we found that over 80% of the M6 cells took up and expressed the beta-gal gene with a cell survival rate of about 50%. In contrast, the transfection efficiency was less than 20% when the M6 cells were electroporated in suspension. It was shown that CV-1 cells could also be electroporated highly efficiently using the in situ method. Furthermore, we have measured the time required to express the beta-gal gene after the plasmid DNA was introduced. We found that the percentage of cells expressing beta-gal reached a peak value about 10 h after electroporation. This time-course was the same for both attached and suspended cells, suggesting that the observed difference in transfection efficiency was mainly the result of effects of the detachment treatment on the electroporation process rather than on the gene expression.     

Simple and effective method of electroporation for introduction of plasmid and cosmid DNAs to mammalian cells

We have established a simple and efficient method of electroporation applicable to gene transfer in mammalian cells. It uses a single decaying pulse of around 1 ms at room temperature in the medium such as Saline G appropriate for repair of pulse-induced pores in the plasma membrane. Many types of cells (both floating and adherent) could be transformed efficiently by the electric field strengths between 1-2 kV/cm. For instance P3U1, mouse myeloma cell, could be transformed by a pulse at 1.2 kV/cm with the frequency of 10(-2) per viable cells and with survivals of 90%. We have applied these conditions to transform tsBN2 cell line of BHK21/13 by a cosmid clone (approximately 45 kb) carrying the human gene complementing to tsBN2 mutation. Significant levels of transformation were observed for this gene. Since this gene can only work as a whole size (approximately 30 kb), the results show that electroporation is useful to introduce cosmid or possibly genomic DNA to mammalian cells.     

Apoptosis induction with electric pulses — A new approach to cancer therapy with drug free

Electrical pulses have been widely used in biomedical fields, whose applications depend on the parameters such as durations and electric intensity. Conventional electroporation (0.1-1 kV/cm, 100 μs) has been used in cell fusion, transfection and electrochemotherapy. Recent studies with high-intensity (MV/cm) electric field applications with durations of several tens of nanoseconds can affect intracellular signal transduction and intracellular structures with plasma intact, resulting in an application of intracellular manipulation. The most recent development is the finding that parameters between those two ranges could be used to induce apoptosis of cancer cells. Proposal of apoptosis induction and tumor inhibition has advantages to pursue the treatment of cancer free of cytotoxic drugs.     

Diverse effects of nanosecond pulsed electric fields on cells and tissues

The application of pulsed electric fields to cells is extended to include nonthermal pulses with shorter durations (10-300 ns), higher electric fields (< or =350 kV/cm), higher power (gigawatts), and distinct effects (nsPEF) compared to classical electroporation. Here we define effects and explore potential application for nsPEF in biology and medicine. As the pulse duration is decreased below the plasma membrane charging time constant, plasma membrane effects decrease and intracellular effects predominate. NsPEFs induced apoptosis and caspase activation that was calcium-dependent (Jurkat cells) and calcium-independent (HL-60 and Jurkat cells). In mouse B10-2 fibrosarcoma tumors, nsPEFs induced caspase activation and DNA fragmentation ex vivo, and reduced tumor size in vivo. With conditions below thresholds for classical electroporation and apoptosis, nsPEF induced calcium release from intracellular stores and subsequent calcium influx through store-operated channels in the plasma membrane that mimicked purinergic receptor-mediated calcium mobilization. When nsPEF were applied after classical electroporation pulses, GFP reporter gene expression was enhanced above that observed for classical electroporation. These findings indicate that nsPEF extend classical electroporation to include events that primarily affect intracellular structures and functions. Potential applications for nsPEF include inducing apoptosis in cells and tumors, probing signal transduction mechanisms that determine cell fate, and enhancing gene expression.     

Effects of pulsed electric fields on mouse spleen lymphocytes in vitro

The effects of pulsed electric fields on cell membranes were investigated. In vitro exposure of mouse splenocytes to a single high-voltage pulse resulted in an increase in membrane permeability that was dependent on both the electric field strength and the pulse duration. Exposure to a 2 μs, 3.0 kV/cm pulse resulted in the induction of a 1.26 V transmembrane potential, and elicited a 50% loss of intracellular K⁺. These results are in agreement with previous studies of the effects of pulsed electric fields on erythrocytes and microorganisms. The effect of pulsed electric fields on the functional integrity of lymphocytes was i vestigated by measuring [³H]thymidine incorporation by cells cultured in the presence and absence of various mitogens following exposure to an electrical pulse. No statistically significant effects on the response of mouse spleen lymphocytes to concanavalin A, phytohemagglutinin or lipopolysaccharide were observed following exposure to 2 μs electric pulses at amplitudes of up to 3.5 kV/cm. Exposure to a single 10 μs pulse of 2.4–3.5 kV/cm produced a statistically significant reduction in the response of lymphocytes to lipopolysaccharide stimulation that was attributed to cell death.     

Permeabilization of tumor cells induced by pulsed electric fields in vitro

The permeabilization of tumor cells in vitro under the action of pulsed electric fields with a duration of 6 mks in the range of amplitudes 1-7 kV/cm was studied. In the mode of excitation in the ambience of localized plasma discharge in a chamber of special design, an enhanced damage to cells in suspension was observed. It is assumed that the enhancement is due to the synchronous action of the electric field and acoustic shock wave pulses. In the mode without the plasma breakdown of ambience, when the pulse duration of electric field of intensity of 1-2 kV/cm was increased to 60 mks, the efficiency of permeabilization increases nearly by one order. The experimental results are compared with the known theoretical models of cell membrane electroporation.     

Electroporation optimization to deliver plasmid DNA into dental follicle cells

Electroporation is a simple and versatile approach for DNA transfer but needs to be optimized for specific cells. We conducted square wave electroporation experiments for rat dental follicle cells under various conditions. These experiments indicated that the optimal electroporation electric field strength was 375 V/cm, and that plasmid concentrations greater than 0.18 μg/μL were required to achieve high transfection efficiency. BSA or fetal bovine serum in the pulsing buffer significantly improved cell survival and increased the number of transfected cells. The optimal pulsing duration was in the range of 45–120 ms at 375 V/cm. This electroporation protocol can be used to deliver DNA into dental follicle cells to study the roles of candidate genes in regulating tooth eruption. This is the first report showing the transfection of dental follicle cells using electroporation. The parameters determined in this study are likely to be applied to transfection of other fibroblast cells.     

Optimization of an electroporation protocol using the K562 cell line as a model: role of cell cycle phase and cytoplasmic DNAses

The improvement of gene therapy protocols is intimately related to the establishment of efficient gene transfer methods. Electroporation has been increasingly employed in in vitro and in vivo protocols, and much attention has been given to increasing its transfection potential. The method is based on the application of an electric field of short duration and high voltage to the cells, forming reversible pores through which molecules can enter the cell. In this work, we describe the optimization of a protocol for the electroporation of K562 cells involving the combination of electric field, resistance and capacitance values. Using RPMI 1640 as pulsing buffer and 30 μg of pEGFP-N1 plasmid, 875 V cm⁻¹, 500 μF and infinite resistance, we achieved transfection rates of 82.41 ± 3.03%, with 62.89 ± 2.93% cell viability, values higher than those reported in the literature. Analyzing cell cycle after electroporation, with three different electric field conditions, we observed that in a heterogeneous population of cells, viability of G₁ cells is less affected by electroporation than that of cells in late S and G₂/M phases. We also observed that efficiency of electroporation can be improved using the DNAse inhibitor Zn, immediately after the pulse. These results can represent a significant improvement of current methods of electroporation of animal and plant cells.     

Conceptual design of integrated microfluidic system for magnetic cell separation,electroporation, and transfection

For the purposes of a successful ex vivo gene therapy we have proposed and analyzed a new concept of an integrated microfluidic system for combined magnetic cell separation, electroporation, and magnetofection. For the analysis of magnetic and electric field distribution (given by Maxwell equations) as well as dynamics of magnetically labeled cell and transfection complex, we have used finite element method directly interfaced to the Matlab routine solving Newton dynamical equations of motion. Microfluidic chamber has been modeled as a channel with height and length 1 mm and 1 cm, respectively. Bottom electrode consisted of 100 parallel ferromagnetic straps and the upper electrode was plate of diamagnetic copper. From the dynamics of magnetic particle motion we have found that the characteristic time-scales for the motion of cells (mean capture time ～ 4 s) and gene complexes (mean capture time ～ 3 min), when permanent magnets are used, are in the range suitable for efficient cell separation and gene delivery. The largest electric field intensity (～10 kV/m) was observed at the edges of the microelectrodes, in the close proximity of magnetically separated cells, which is optimal for subsequent cell electroporation.     

Gene transfer into mouse lyoma cells by electroporation in high electric fields.

Electric impulses (8 kV/cm, 5 microseconds) were found to increase greatly the uptake of DNA into cells. When linear or circular plasmid DNA containing the herpes simplex thymidine kinase (TK) gene is added to a suspension of mouse L cells deficient in the TK gene and the cells are then exposed to electric fields, stable transformants are formed that survive in the HAT selection medium. At 20 degrees C after the application of three successive electric impulses followed by 10 min to allow DNA entry there result 95 (+/- 3) transformants per 10(6) cells and per 1.2 micrograms DNA. Compared with biochemical techniques, the electric field method of gene transfer is very simple, easily applicable, and very efficient. Because the mechanism of DNA transport through cell membranes is not known, a simple physical model for the enhanced DNA penetration into cells in high electric fields is proposed. According to this ' electroporation model' the interaction of the external electric field with the lipid dipoles of a pore configuration induces and stabilizes the permeation sites and thus enhances cross membrane transport.     

Novel Parallelized Electroporation by Electrostatic Manipulation of a Water-in-Oil Droplet as a Microreactor

Electroporation is the most widely used transfection method for delivery of cell-impermeable molecules into cells. We developed a novel gene transfection method, water-in-oil (W/O) droplet electroporation, using dielectric oil and an aqueous droplet containing mammalian cells and transgene DNA. When a liquid droplet suspended between a pair of electrodes in dielectric oil is exposed to a DC electric field, the droplet moves between the pair of electrodes periodically and droplet deformation occurs under the intense DC electric field. During electrostatic manipulation of the droplet, the local intense electric field and instantaneous short circuit via the droplet due to droplet deformation facilitate gene transfection. This method has several advantages over conventional transfection techniques, including co-transfection of multiple transgene DNAs into even as few as 10³ cells, transfection into differentiated neural cells, and the capable establishment of stable cell lines. In addition, there have been improvements in W/O droplet electroporation electrodes for disposable 96-well plates making them suitable for concurrent performance without thermal loading by a DC electric field. This technique will lead to the development of cell transfection methods for novel regenerative medicine and gene therapy.     

Nanosecond Electric Pulse Effects on Gene Expression

Gene electrotransfection using micro- or millisecond electric pulses is a well-established method for safe gene transfer. For efficient transfection, plasmid DNA has to reach the nucleus. Shorter, high-intensity nanosecond electric pulses (nsEPs) affect internal cell membranes and may contribute to an increased uptake of plasmid by the nucleus. In our study, nsEPs were applied to Chinese hamster ovary (CHO) cells after classical gene electrotransfer, using micro- or millisecond pulses with a plasmid coding the green fluorescent protein (GFP). Time gaps between classical gene electrotransfer and nsEPs were varied (0.5, 2, 6 and 24 h) and three different nsEP parameters were used: 18 ns-10 kV/cm, 10 ns-40 kV/cm and 15 ns-60 kV/cm. Results analyzed by either fluorescence microscopy or flow cytometry showed that neither the percentage of electrotransfected cells nor the amount of GFP expressed was increased by nsEP. All nsEP parameters also had no effects on GFP fluorescence intensity of human colorectal tumor cells (HCT-116) with constitutive expression of GFP. We thus conclude that nsEPs have no major contribution to gene electrotransfer in CHO cells and no effect on constitutive GFP expression in HCT-116 cells.     

Optimization of transfection methods for Huh-7 and Vero cells: A comparative study

Availability of an efficient transfection protocol is the first determinant in success of gene transferring studies in mammalian cells which is accomplished experimentally for every single cell type. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for Huh-7 and Vero cells. Different cell confluencies, DNA/reagent ratios and total transfection volumes were optimized for two chemical reagents including jetPEI? and Lipofectamine? 2000. Besides, the effects of electric field strength and pulse length were investigated to improve electroporation efficiency. Transfection of cells by pEGFP-N1 vector and tracking the expression of GFP by FACS and Fluorescence Microscopy analysis were the employed methods to evaluate transfection efficiencies. Optimized electroporation protocols yielded 63.73 ± 2.36 and 73.9 ± 1.6% of transfection in Huh-7 and Vero cells respectively, while maximum achieved level of transfection by jetPEI? was 14.2 ± 0.69 and 28 ± 1.11% Huh-7 and Vero cells, respectively. Post transfectional chilling of the cells did not improve electrotransfection efficiency of Huh-7 cells. Compared to chemical based reagents, electroporation showed superior levels of transfection in both cell lines. The presented protocols should satisfy most of the experimental applications requiring high transfection efficiencies of these two cell lines.     

High efficiency,Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes^1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies^9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies¹³. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells^14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies^{19, 24-26}. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection²⁷. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures^17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.     

Quantification of electroporative uptake kinetics and electric field heterogeneity effects in cells

We have conducted experiments quantitatively investigating electroporative uptake kinetics of a fluorescent plasma membrane integrity indicator, propidium iodide (PI), in HL60 human leukemia cells resulting from exposure to 40 μs pulsed electric fields (PEFs). These experiments were possible through the use of calibrated, real-time fluorescence microscopy and the development of a microcuvette: a specialized device designed for exposing cell cultures to intense PEFs while carrying out real-time microscopy. A finite-element electrostatic simulation was carried out to assess the degree of electric field heterogeneity between the microcuvette's electrodes allowing us to correlate trends in electroporative response to electric field distribution. Analysis of experimental data identified two distinctive electroporative uptake signatures: one characterized by low-level, decelerating uptake beginning immediately after PEF exposure and the other by high-level, accelerating fluorescence that is manifested sometimes hundreds of seconds after PEF exposure. The qualitative nature of these fluorescence signatures was used to isolate the conditions required to induce exclusively transient electroporation and to discuss electropore stability and persistence. A range of electric field strengths resulting in transient electroporation was identified for HL60s under our experimental conditions existing between 1.6 and 2 kV/cm. Quantitative analysis was used to determine that HL60s experiencing transient electroporation internalized between 50 and 125 million nucleic acid-bound PI molecules per cell. Finally, we show that electric field heterogeneity may be used to elicit asymmetric electroporative PI uptake within cell cultures and within individual cells.     

Electroporation and electrophoretic DNA transfer into cells. The effect of DNA interaction with electropores.

It has been shown recently that electrically induced DNA transfer into cells is a fast vectorial process with the same direction as DNA electrophoresis in an external electric field (Klenchin, V. A., S. I. Sukharev, S. M. Serov, L. V. Chernomordik, and Y. A. Chizmadzhev. 1991. Biophys. J. 60:804-811). Here we describe the effect of DNA interaction with membrane electropores and provide additional evidences for the key role of DNA electrophoresis in cell electrotransfection. The assay of electrically induced uptake of fluorescent dextrans (FDs) by cells shows that the presence of DNA in the medium during electroporation leads to a sharp increase in membrane permeability to FDs of M(r) < 20,000. The permeability increases with DNA concentration and the effect is seen even if FD is added to the cell suspension a few minutes after pulse application. The longer the DNA fragment, the greater the increase in permeability. The use of a two-pulse technique allows us to separate two effects provided by a pulsed electric field: membrane electroporation and DNA electrophoresis. The first pulse (6 kV/cm, 10 microseconds) creates pores efficiently, whereas transfection efficiency (TE) is low. The second pulse of much lower amplitude, but substantially longer (0.2 kV/cm, 10 ms), does not cause poration and transfection by itself but enhances TE by about one order of magnitude. In two-pulse experiments, TE rises monotonously with the increase of the second pulse duration. By varying the delay duration between the two pulses, we estimate the lifetime of electropores (which are DNA-permeable in conditions of low electric field) as tens of seconds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrical behavior and pore accumulation in a multicellular model for conventional and supra-electroporation

Extremely large but very short (20 kV/cm, 300 ns) electric field pulses were reported recently to non-thermally destroy melanoma tumors. The stated mechanism for field penetration into cells is pulse characteristic times faster than charge redistribution (displacement currents). Here we use a multicellular model with irregularly shaped, closely spaced cells to show that instead overwhelming pore creation (supra-electroporation) is dominant, with field penetration due to pores (ionic conduction currents) during most of the pulse. Moreover, the model's maximum membrane potential (about 1.2 V) is consistent with recent experimental observations on isolated cells. We also use the model to show that conventional electroporation resulting from 100 microsecond, 1 kV/cm pulses yields a spatially heterogeneous electroporation distribution. In contrast, the melanoma-destroying pulses cause nearly homogeneous electroporation of cells and their nuclear membranes. Electropores can persist for times much longer than the pulses, and are likely to be an important mechanism contributing to cell death.     

In vivo electroporation of skeletal muscle: threshold, efficacy and relation to electric field distribution.

In vivo electroporation is increasingly being used to deliver small molecules as well as DNA to tissues. The aim of this study was to quantitatively investigate in vivo electroporation of skeletal muscle, and to determine the threshold for permeabilization. We designed a quantitative method to study in vivo electroporation, by measuring uptake of (51)Cr-EDTA. As electrode configuration influences electric field (E-field) distribution, we developed a method to calculate this. Electroporation of mouse muscle tissue was investigated using either external plate electrodes or internal needle electrodes placed 4 mm apart, and eight pulses of 99 micros duration at a frequency of 1 Hz. The applied voltage to electrode distance ratio was varied from 0 to 2.0 kV/cm. We found that: (1) the threshold for permeabilization of skeletal muscle tissue using short duration pulses was at an applied voltage to electrode distance ratio of 0.53 kV/cm (+/-0.03 kV/cm), corresponding to an E-field of 0.45 kV/cm; (2) there were two phases in the uptake of (51)Cr-EDTA, the first indicating increasing permeabilization and the second indicating beginning irreversible membrane damage; and (3) the calculated E-field distribution was more homogeneous for plate than for needle electrodes, which was reflected in the experimental results.