High-efficiency gene electrotransfer into skeletal muscle: description and physiological applicability of a new pulse generator

Efficiency and reproducibility of gene electrotransfer depend on the electrical specifications provided by the pulse generator, such as pulse duration, pulse number, pulse frequency, pulse combination, and current intensity. Here, we describe the performances of GET42, a pulse generator specifically designed for gene electrotransfer into skeletal muscle. Expression of beta-galactosidase in the Tibialis anterior muscle of Sprague-Dawley male rats was increased 250-fold by GET42 compared to DNA injection alone. Combination of high and low current intensity pulses further increased transfection efficiency (400-fold compared to DNA injection without electrotransfer). Varying degrees of muscle necrosis were observed after gene electrotransfer. Nevertheless, muscle necrosis was dramatically reduced after optimization of cumulated pulse duration without significant reduction in transfection efficiency. Physiological applicability was illustrated by the analysis of cytochrome c promoter transactivation. In conclusion, GET42 has proven to be a reliable and efficient pulse generator for gene electrotransfer experiments, and provides a powerful mean to study in vivo the regulation of gene expression.     

Long-term,high level in vivo gene expression after electric pulse-mediated gene transfer into skeletal muscle

Gene delivery to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the local or systemic secretion of therapeutic proteins. However, current DNA delivery technologies have to be improved. We report very efficient luciferase gene transfer into muscle fibres obtained through the delivery of squarewave electric pulses of moderate field strength (100–200 V/cm) and of long duration (20 ms) to muscle previously injected with plasmid DNA. This intramuscular ‘electrotransfer’ method increases reporter gene expression by more than 100 times. It is noteworthy that this expression remains high and stable for at least 9 months. Moreover, intramuscular electrotransfer strongly decreases the interindividual variability usually observed after plasmid DNA injection into muscle fibres. Therefore, DNA electrotransfer in muscle possesses broad potential applications in gene therapy and for physiological, pharmacological and developmental studies.     

Influence of Plasmid Concentration on DNA Electrotransfer In Vitro Using High-Voltage and Low-Voltage Pulses

DNA electrotransfer in vivo for gene therapy is a promising method. For further clinical developments, the efficiency of the method should be increased. It has been shown previously that high efficiency of gene electrotransfer in vivo can be achieved using high-voltage (HV) and low-voltage (LV) pulses. In this study we evaluated whether HV and LV pulses could be optimized in vitro for efficient DNA electrotransfer. Experiments were performed using Chinese hamster ovary (CHO) cells. To evaluate the efficiency of DNA electrotransfer, two different plasmids coding for GFP and luciferase were used. For DNA electrotransfer experiments 50 μl of CHO cell suspension containing 100, 10 or 1 μg/ml of the plasmid were placed between plate electrodes and subjected to various combinations of HV and LV pulses. The results showed that at 100 μg/ml plasmid concentration LV pulse delivered after HV pulse increased neither the percentage of transfected cells nor the total transfection efficiency (luciferase activity). The contribution of the LV pulse was evident only at reduced concentration (10 and 1 μg/ml) of the plasmid. In comparison to HV (1,200 V/cm, 100 μs) pulse, addition of LV (100 V/cm, 100 ms) pulse increased transfection efficiency severalfold at 10 μg/ml and fivefold at 1 μg/ml. At 10 μg/ml concentration of plasmid, application of four LV pulses after HV pulse increased transfection efficiency by almost 10-fold. Thus, these results show that contribution of electrophoretic forces to DNA electrotransfer can be investigated in vitro using HV and LV pulses.     

In vivo gene electrotransfer into skeletal muscle: effects of plasmid DNA on the occurrence and extent of muscle damage

BACKGROUND: Understanding the mechanisms underlying gene electrotransfer muscle damage can help to design more effective gene electrotransfer strategies for physiological and therapeutical applications. The present study investigates the factors involved in gene electrotransfer associated muscle damage. METHODS: Histochemical analyses were used to determine the extent of transfection efficiency and muscle damage in the Tibialis anterior muscles of Sprague-Dawley male rats after gene electrotransfer. RESULTS: Five days after gene electrotransfer, features of muscle degeneration and regeneration were consistently observed, thus limiting the extent of transfection efficiency. Signs of muscle degeneration/regeneration were no longer evident 21 days after gene electrotransfer except for the presence of central myonuclei. Neither the application of electrical pulses per se nor the extracellular presence of plasmid DNA per se contributed significantly to muscle damage (2.9 +/- 1.0 and 2.1 +/- 0.7% of the whole muscle cross-sectional area, respectively). Gene electrotransfer of a plasmid DNA, which does not support gene expression, increased significantly muscle damage (8.7 +/- 1.2%). When plasmid DNA expression was permitted (gene electrotransfer of pCMV-beta-galactosidase), muscle damage was further increased to 19.7 +/- 4.5%. Optimization of cumulated pulse duration and current intensity dramatically reduced gene electrotransfer associated muscle damage. Finally, mathematical modeling of gene electrotransfer associated muscle damage as a function of the number of electrons delivered to the tissue indicated that pulse length critically determined the extent of muscle damage. CONCLUSION: Our data suggest that neither the extracellular presence of plasmid DNA per se nor the application of electric pulses per se contributes significantly to muscle damage. Gene electrotransfer associated muscle damage mainly arises from the intracellular presence and expression of plasmid DNA.     

Nucleic Acids Electrotransfer-Based Gene Therapy (Electrogenetherapy): Past,Current, and Future

About 25 years after the publication of the first report on gene transfer in vitro in cultured cells by the means of electric pulses delivery, reversible cell electroporation for gene transfer and gene therapy (DNA electrotransfer) is at a cross in its development. Present knowledge on the effects of cell exposure to appropriate electric field pulses, particularly at the level of the cell membrane, is reported here. The importance of the models of electric field distribution in tissues and of the correct choice of electrodes and applied voltages is highlighted. The mechanisms involved in DNA electrotransfer, which include cell electropermeabilization and DNA electrophoresis, are also surveyed. This knowledge has allowed developing new nucleic acids electrotransfer conditions using combinations of permeabilizing pulses of high voltage and short duration, and of electrophoretic pulses of low voltage and long duration, which are very efficient and safer. Feasibility of electric pulses delivery for gene transfer in humans is discussed taking into account that electric pulses delivery is already regularly used for localized drug delivery in the treatment of cutaneous and subcutaneous solid tumors by electrochemotherapy. Because recent technological developments made DNA electrotransfer more and more efficient and safer, this non-viral gene therapy approach is now ready to reach the clinical stage. A good understanding of DNA electrotransfer principles and the respect of safe procedures will be key elements for a successful future transfer DNA electrotransfer into the clinics.     

Optimization of Electric Pulse Amplitude and Frequency In Vitro for Low Voltage and High Frequency Electrochemotherapy

During standard electrochemotherapy (ECT), using a train of 1,000 V/cm amplitude rectangular pulses with 1 Hz frequency, patients experience an unpleasant sensation and slight edema. According to the patients, muscle contractions provoked by high amplitude (about 1,000 V/cm) and low repetition frequency (1 Hz) pulses are the most unpleasant and painful sensations. Recently, ECT using low voltage and higher repetition frequency (LVHF) has been shown to be an effective tool for inhibiting tumor growth. The aim of the present study was to optimize electric pulse amplitude and repetition frequency for LVHF ECT by sampling the different sets of pulse parameters on cell viability and permeabilization. In ECT, a reversible effect based on high permeabilization is desirable. For this purpose, we used bleomycin to evaluate the permeabilization of K562 and MIA-PACA2 cells caused by low voltage (50–150 V/cm) and higher repetition frequency (4–6 kHz) electric pulses. We show that the reversible effect with electropermeabilization of the cells caused by LVHF ECT is accessible; this interaction is more effective for electric pulses with 70 V/cm amplitude.     

Electrotransfer of human IL-1Ra into skeletal muscles reduces the incidence of murine collagen-induced arthritis

BACKGROUND: It has previously been demonstrated that high levels of gene expression in skeletal muscles can be achieved after direct in vivo electrotransfer of naked plasmid DNA. The purpose of this study is to examine the potential of in vivo electroporation of plasmid DNA encoding human IL-1Ra for the prevention of murine collagen-induced arthritis (CIA). METHODS: DBA/1 mice were injected in gastrocnemius muscles with plasmid DNA followed by in vivo electroporation. To uncover the optimum conditions of gene transfer, various electric field strengths and different amounts of plasmid DNA were applied. Calf muscles around the injected areas were investigated with histological methods for damage to muscle tissue. The levels of human IL-1Ra expression in the injected area and also in the serum were determined with ELISA for human IL-1Ra. Based on these data, the effects of electrotransfer of plasmid DNA were tested using the murine CIA model. DBA/1 mice were immunized with bovine collagen type II at the base of the tail. On day 21, mice were given a booster injection with the same antigen. Mice were divided into two groups on day 26. One group of mice received plasmid containing the IL-1Ra cDNA sequence, while control mice were given plasmid lacking the IL-1Ra coding sequence. The incidence of arthritis was evaluated by macroscopic analysis, histological analysis, and the levels of inflammatory cytokines. RESULTS: IL-1Ra expression increased as a function of the electrical field strength and the amount of DNA. 200 V/cm (eight pulses; 20 ms per pulse; 1 Hz) and 15 microg of plasmid DNA per mouse were found to be optimum for gene transfer. After in vivo electroporation, gene expression in both muscle and serum increased gradually, reaching a peak value on day 10. Significant levels of human IL-1Ra expression were maintained for 20 days. Macroscopic analysis showed that the onset of CIA was significantly inhibited by direct electrotransfer of plasmid DNA encoding human IL-1Ra. Histological analysis of knee joints showed that the incidence of arthritis in knee joints was also prevented. The levels of mouse IL-1beta and IL-12 in paws were significantly lower in the group treated with IL-1Ra than those in the control group. CONCLUSIONS: These results demonstrate that direct electrotransfer of plasmid containing the human IL-1Ra cDNA sequence to skeletal muscle can reduce the incidence of CIA in mice.     

Use of Collagen Gel as a Three-Dimensional In Vitro Model to Study Electropermeabilization and Gene Electrotransfer

Gene electrotransfer is a promising nonviral method that enables transfer of plasmid DNA into cells with electric pulses. Although many in vitro and in vivo studies have been performed, the question of the implied gene electrotransfer mechanisms is largely open. The main obstacle toward efficient gene electrotransfer in vivo is relatively poor mobility of DNA in tissues. Since cells are mechanically coupled to their extracellular environment and act differently compared to standard in vitro conditions, we developed a three-dimensional (3-D) in vitro model of CHO cells embedded in collagen gel as an ex vivo model of tissue to study electropermeabilization and different parameters of gene electrotransfer. For this purpose, we first used propidium iodide to detect electropermeabilization of CHO cells embedded in collagen gel. Then, we analyzed the influence of different concentrations of plasmid DNA and pulse duration on gene electrotransfer efficiency. Our results revealed that even if cells in collagen gel can be efficiently electropermeabilized, gene expression is significantly lower. Gene electrotransfer efficiency in our 3-D in vitro model had similar dependence on concentration of plasmid DNA and pulse duration comparable to in vivo studies, where longer (millisecond) pulses were shown to be more optimal compared to shorter (microsecond) pulses. The presented results demonstrate that our 3-D in vitro model resembles the in vivo situation more closely than conventional 2-D cell cultures and, thus, provides an environment closer to in vivo conditions to study mechanisms of gene electrotransfer.     

The Role of Electrophoresis in Gene Electrotransfer

Gene electrotransfer is an established method for gene delivery which uses high-voltage pulses to increase the permeability of a cell membrane and enables transfer of genes. Poor plasmid mobility in tissues is one of the major barriers for the successful use of gene electrotransfer in gene therapy. Therefore, we analyzed the effect of electrophoresis on increasing gene electrotransfer efficiency using different combinations of high-voltage (HV) and low-voltage (LV) pulses in vitro on CHO cells. We designed a special prototype of electroporator, which enabled us to use only HV pulses or combinations of LV + HV and HV + LV pulses. We used optimal plasmid concentrations used in in vitro conditions as well as lower suboptimal concentrations in order to mimic in vivo conditions. Only for the lowest plasmid concentration did the electrophoretic force of the LV pulse added to the HV pulse increase the transfection efficiency compared to using only HV. The effect of the LV pulse was more pronounced for HV + LV, while for the reversed sequence, LV + HV, there was only a minor effect of the LV pulse. For the highest plasmid concentrations no added effect of LV pulses were observed. Our results suggest that there are different contributing effects of LV pulses: electrophoretically increased contact of DNA with the membrane and increased insertion of DNA into permeabilized cell membrane and/or translocation due to electrophoretic force, which appears to be the dominant effect.     

Calcium influx determines the muscular response to electrotransfer

Cell membrane permeabilization by electric pulses (electropermeabilization), results in free exchange of ions across the cell membrane. The role of electrotransfer-mediated Ca(2+)-influx on muscle signaling pathways involved in degeneration (β-actin and MurF), inflammation (IL-6 and TNF-α), and regeneration (MyoD1, myogenin, and Myf5) was investigated, using pulse parameters of both electrochemotherapy (8 HV) and DNA delivery (HVLV). Three pulsing conditions were used: 8 high-voltage pulses (8 HV), resulting in large permeabilization and ion flux, and a combination of one high-voltage pulse and one low-voltage pulse (HVLV), either alone or in combination with injection of DNA. Mice and rats were anesthetized before pulsing. At the times given, animals were killed, and intact tibialis cranialis muscles were excised for analysis. Uptake of Ca(2+) was assessed using (45)Ca as a tracer. Using gene expression analyses and histology, we showed a clear association between Ca(2+) influx and muscular response. Moderate Ca(2+) influx induced by HVLV pulses results in activation of pathways involved in immediate repair and hypertrophy. This response could be attenuated by intramuscular injection of EGTA reducing Ca(2+) influx. Larger Ca(2+) influx as induced by 8-HV pulses leads to muscle damage and muscle fiber regeneration through recruitment of satellite cells. The extent of Ca(2+) influx determines the muscular response to electrotransfer and, thus, the success of a given application. In the case of electrochemotherapy, in which the objective is cell death, a large influx of Ca(2+) may be beneficial, whereas for DNA electrotransfer, muscle recovery should occur without myofiber loss to ensure preservation of plasmid DNA.     

Numerical optimization of gene electrotransfer into muscle tissue

Background  

Electroporation-based gene therapy and DNA vaccination are promising medical applications that depend on transfer of pDNA into target tissues with use of electric pulses. Gene electrotransfer efficiency depends on electrode configuration and electric pulse parameters, which determine the electric field distribution. Numerical modeling represents a fast and convenient method for optimization of gene electrotransfer parameters. We used numerical modeling, parameterization and numerical optimization to determine the optimum parameters for gene electrotransfer in muscle tissue.     

Plasmid DNA electrotransfer for intracellular and secreted proteins expression: new methodological developments and applications

In vivo electrotransfer is a physical method of gene delivery in various tissues and organs, relying on the injection of a plasmid DNA followed by electric pulse delivery. The importance of the association between cell permeabilization and DNA electrophoresis for electrotransfer efficiency has been highlighted. In vivo electrotransfer is of special interest since it is the most efficient non-viral strategy of gene delivery and also because of its low cost, easiness of realization and safety. The potentiality of this technique can be further improved by optimizing plasmid biodistribution in the targeted organ, plasmid structure, and the design of the encoded protein. In particular, we found that plasmids of smaller size were electrotransferred more efficiently than large plasmids. It is also of importance to study and understand kinetic expression of the transgene, which can be very variable, depending on many factors including cellular localization of the protein, physiological activity and regulation. The most widely targeted tissue is skeletal muscle, because this strategy is not only promising for the treatment of muscle disorders, but also for the systemic secretion of therapeutic proteins. Vaccination and oncology gene therapy are also major fields of application of electrotransfer, whereas application to other organs such as liver, brain and cornea are expanding. Many published studies have shown that plasmid electrotransfer can lead to long-lasting therapeutic effects in various pathologies such as cancer, blood disorders, rheumatoid arthritis or muscle ischemia. DNA electrotransfer is also a powerful laboratory tool to study gene function in a given tissue.     

Effect of electric field vectoriality on electrically mediated gene delivery in mammalian cells

Electropermeabilization is a nonviral method used to transfer genes into living cells. Up to now, the mechanism is still to be elucidated. Since cell permeabilization, a prerequired for gene transfection, is triggerred by electric field, its characteristics should depend on its vectorial properties. The present investigation addresses the effect of pulse polarity and orientation on membrane permeabilization and gene delivery by electric pulses applied to cultured mammalian cells. This has been directly observed at the single-cell level by using digitized fluorescence microscopy. While cell permeabilization is only slightly affected by reversing the polarity of the electric pulses or by changing the orientation of pulses, transfection level increases are observed. These last effects are due to an increase in the cell membrane area where DNA interacts. Fluorescently labelled plasmids only interact with the electropermeabilized side of the cell facing the cathode. The plasmid interaction with the electropermeabilized cell surface is stable and is not affected by pulses of reversed polarities. Under such conditions, DNA interacts with the two sites of the cell facing the two electrodes. When changing both the pulse polarity and their direction, DNA interacts with the whole membrane cell surface. This is associated with a huge increase in gene expression. This present study demonstrates the relationship between the DNA/membrane surface interaction and the gene transfer efficiency, and it allows to define the experimental conditions to optimize the yield of transfection of mammalian cells.     

Delivery of electric pulses for DNA electrotransfer to mouse muscle does not induce the expression of stress related genes

In vivo gene transfer to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic delivery of therapeutic proteins. Electrotransfer is a powerful method for DNA transfer into skeletal muscle. In view of the broad potential gene therapy clinical application of electrotransfer offers, it is important to perform toxicology studies on electrotransfered muscle tissue. We have investigated if the delivery of square wave electric pulses of low field strength and long duration to mouse tibial cranial muscle induced the expression of stress related genes. We have profiled gene expression patterns in muscles at different times after delivery of electric pulses using Stress/Toxicology microarrays. No significant variation in the expression of stress related-genes was detected between treated and non-treated muscles. This suggests that application of adequate, fine-tuned, electric pulses to the skeletal muscle is a non-toxic technique for gene therapy.     

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.     

Muscle transfection and permeabilization induced by electrotransfer or pluronic® L64: Paired study by optical imaging and MRI

Background

Muscle transfection by electrotranfer is an efficient currently used procedure. Recently, the block copolymer pluronic L64 has been reported to improve muscle transfection. Both procedures are known to permeabilize muscle fibres. Relation between muscle transfection and permeabilization by electrotransfer and L64 was investigated herein.

Methods

Muscle transfection was evaluated by optical detection of the luciferase reporter gene activity. Muscle permeabilization was evaluated by the uptake of the T1 contrast agent gadolinium-Dotarem (Gd-DOTA) using Magnetic resonance imaging (MRI). Histological examination of muscle sections was also performed.

Results

Electrotransfer and L64 (at a 0.25% concentration) similarly improved muscle transfection, although the interindividual variability was higher for pluronic. On the same animals, the permeabilized volume to the Gd-DOTA was significantly increased after electrotransfer, and L64 from 0.1% to 1%. The concentration of the Gd-DOTA in the permeabilized volume was significantly increased after electrotransfer and L64 at 0.5% and 1%. By histological observation, the inflammation was maximum at day 3 after electrotransfer or L64 injection, and mostly reversed after 7 days. The permeabilized volume and the transfection level correlated for the set of all the conditions tested. However, no significant correlation was observed between Gd-DOTA concentration and transfection.

General significance

It is possible to use successively on the same animals MRI and optical imaging for paired studies of muscle transfection and permeabilization. Permeabilization is possibly not related to gene transfer but it indicates membrane modification related to transfection by the electrotransfer or co-injection of DNA with the L64.     

Electroporation of human embryonic stem cells: Small and macromolecule loading and DNA transfection

Cryopreservation, directed differentiation, and genetic manipulation of human embryonic stem cells (hESCs) all require the transport of exogenous small molecules, proteins, or DNA into the cell. The absence of standard small and macromolecule loading techniques in hESCs as well as the inadequacies of current DNA transfection techniques have led us to develop electroporation as an efficient loading and transfection methodology. The electroporation parameters of pulse voltage, duration, and number have been explored and evaluated in terms of cell viability, molecular loading, and transfection efficiency on a per cell basis. Small molecule loading was assessed using propidium iodide (PI) and the disaccharide trehalose. Additionally, protein loading was investigated using a glutathione-S-transferase green fluorescent protein (GST-GFP) conjugate, and DNA transfection optimization was performed by constitutive expression of GFP from a plasmid. The optimum pulse voltage must balance cell viability, which decreases as voltage increases, and loading efficiency, which increases at higher voltages. Short pulse times of 0.05 ms facilitated PI and trehalose loading, whereas 0.5 ms or more was required for GST-GFP loading and DNA transfection. Multiple pulses increased per cell loading of all molecules, though there was a dramatic loss of viability with GST-GFP loading and DNA transfection, likely resulting from the longer pulse duration required to load these molecules.     

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)     

Immune response and inhibition of bacterial growth by electrotransfer of plasmid DNA containing the antimicrobial peptide,epinecidin-1, into zebrafish muscle

Bacterial infections represent serious diseases in aquaculture, rapidly leading to fish death by septicemia. We investigated whether the electrotransfer of green fluorescent protein gene fusion epinecidin-1 (CMV-gfp-epi) DNA into zebrafish muscle could regulate the fish immune response and inhibit bacterial growth. Electroporation parameters such as the number of pulses, voltage, and amount of plasmid DNA were analyzed, and results demonstrated the greatest mRNA expression level of gfp-epi relative to β-actin was obtained with a pulse number of 4, a voltage strength of 100 V/cm, a concentration of DNA of 90 μg/fish, and electroporation for 96 h. In addition, the cytomegalovirus (CMV) promoter exhibited higher activity compared to the mylz promoter in muscle for electrotransfer in zebrafish. GFP fluorescence and gfp-epi mRNA expression in tissues after electroporation were also studied by a polymerase chain reaction, immunohistochemistry, and fluorescence microscopy. gfp-epi expression was significantly correlated with decreased bacterial numbers and immune-related gene expression. These data demonstrate that electroporation of epinecidin-1 might have provoked an inflammatory response that accounts for the improvement in bacterial clearance.