Electric Pulse Parameters Research Articles

3:36 PM Abstract No. 194 Irreversible electroporation can selectively ablate cancer cells without affecting proliferation or effector function of chimeric antigen receptor engineered T cells

We investigated whether differences in cell morphology and size can enable irreversible electroporation (IRE) to have greater cytotoxicity to cancer cells than Chimeric Antigen Receptor (CAR) T cells. IRE was performed on murine (AE17) and human cancer cell lines (MGM and MSTO), and CAR T cells by varying applied voltage, pulse width and pulse numbers in 4-mm gap cuvettes. Treatment parameters showing greatest differential cytotoxicity between cancer and CAR T cells was further tested with IRE performed in a 3D tumor mimic. Confocal microscopy with fluorescent staining for cytoplasm, nucleus, cell death and viability were used to extract cell morphology and spatial extent of IRE. Cell survival and proliferation was quantified at 24 hours post-IRE for all studies. Function of IRE treated, and control CAR T cells was compared with a 51Cr release assay at increasing effector to target ratio. Comsol simulations were used to model electric field gradient in tumor mimic, induced transmembrane voltage for different cell types, and statistical probability of cell death from IRE. Simulations models reconstructed using confocal imaging indicated that cancer cells develop a transmembrane potential that is 1.5 – 2-fold higher than T cells when exposed to the same electric field, increasing their susceptibility for membrane permeabilization. Pulse parameter (1000 V/cm, 20 μs pulse width, 200 pulses) yielding maximum differential cytotoxicity (MSTO: 1.7±0.7% vs. CAR T cell: 61.8 ± 6.6%, P < 0.01) was validated in the 3D tumor mimic (MSTO: 52% vs. CAR T cell: 88% viability on flow cytometry), where we found IRE did not alter the proliferation of CAR T cells or effector function measured with the 51Cr release assay (p not significant). Simulation models of 3D tumor mimic allowed estimation of electric field gradient in vitro along with estimation of relative cell viability as a function of distance from the electrodes based on the statistical models. Simulation-guided and experimental optimization of electric pulse parameters enable IRE with greater cytotoxicity to cancer cells than CAR T cells, increasing the effector to target ratio in the tumor microenvironment.

Nanosecond electric pulses rapidly enhance the inactivation of Gram-negative bacteria using Gram-positive antibiotics

Physically disrupting microorganism membranes to enable antibiotics to overcome resistance mechanisms that inhibit or excrete antibiotics has great potential for reducing antibiotic doses and rendering resistance mechanisms inert. We demonstrate the synergistic inactivation of a Gram-positive (Staphylococcus aureus) and two Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria by combining 222 30kV/cm electric pulses (EPs) or 500 20kV/cm EPs with 300-ns EP duration with various antibiotics with different mechanisms of action is demonstrated. Doses of antibiotics that produced no inactivation in 10min of exposure in solution with bacteria induced several log reductions under the influence of nanosecond EPs. Combining 2μg/L or 20μg/mL of rifampicin with the 30kV/cm EPs enhanced Staphylococcus aureus inactivation compared with EPs alone, while only a few of the other combinations demonstrated improvement. Combining 2μg/L or 20μg/mL of mupirocin or rifampicin with either EP train enhanced E. coli inactivation compared with EPs alone. Combining 2μg/L or 20μg/mL of erythromycin or vancomycin with the 30kV/cm EPs enhanced E. coli inactivation compared with EPs alone. These results indicate that EPs can make Gram-positive antibiotics efficient for inactivating Gram-negative bacteria with future studies required to optimize EP parameters for other antibiotics and Gram-negative bacteria.

Conductivity change with needle electrode during high frequency irreversible electroporation: a finite element study

Abstract Irreversible electroporation (IRE) is a process in which the cell membrane is damaged and leads to cell death. IRE has been used as a minimally invasive ablation tool. This process is affected by some factors. The most important factor is the electric field distribution inside the tissue. The electric field distribution depends on the electric pulse parameters and tissue properties, such as the electrical conductivity of tissue. The present study focuses on evaluating the tissue conductivity change due to high-frequency and low-voltage (HFLV) as well as low-frequency and high-voltage (LFHV) pulses during irreversible electroporation. We were used finite element analysis software, COMSOL Multiphysics 5.0, to calculate the conductivity change of the liver tissue. The HFLV pulses in this study involved 4000 bipolar and monopolar pulses with a frequency of 5 kHz, pulse width of 100 µs, and electric field intensity from 100 to 300 V/cm. On the other hand, the LFHV pulses, which we were used, included 8 bipolar and monopolar pulses with a frequency of 1 Hz, the pulse width of 2 ms and electric field intensity of 2500 V/cm. The results demonstrate that the conductivity change for LFHV pulses due to the greater electric field intensity was higher than for HFLV pulses. The most significant conclusion is the HFLV pulses can change tissue conductivity only in the vicinity of the tip of electrodes. While LFHV pulses change the electrical conductivity significantly in the tissue of between electrodes.

Electric Pulse Pretreatment for Enhanced Lipid Recovery from Chlorella protothecoides

Developing effective, energy-efficient methods to extract lipids from microalgae remains a challenge in bioenergy. This study assesses electric pulse (EP) pretreatments to enhance lipid extraction from Chlorella protothecoides. In tandem with solvent extraction, we applied 10, 50, 100, 200, and 300 60 kV/cm, 60 ns duration EPs or 3, 17, 33, 67, and 100 60 kV/cm, 300 ns duration EPs to match the energy density u applied to the sample for each EP duration. Lipid recovery increased with increasing u for each EP duration until reaching a maximum for 17 300 ns EPs (the second lowest u) and 100 60 ns EPs (the third lowest u) to achieve a 13.3% and 16.1% increase in lipid extraction, respectively. Lipid extraction declined at higher u due to increased cell lysis. The largest net energy released calculated from the difference between the energy available from the extracted lipids and the applied energy of the EPs occurred at the lowest u for each EP duration. Applying trains of 100 μs EPs induced qualitatively similar behavior. These results suggest that a more detailed parametric study may optimize EP parameters for net energy release for specific microalgae and solvents.

Irreversible electroporation for catheter-based cardiac ablation: a systematic review of the preclinical experience.

Irreversible electroporation (IRE) utilizing high voltage pulses is an emerging strategy for catheter-based cardiac ablation with considerable growth in the preclinical arena. A systematic search for articles was performed from three sources (PubMed, EMBASE, and Google Scholar). The primary outcome was the efficacy of tissue ablation with characteristics of lesion formation evaluated by histologic analysis. The secondary outcome was focused on safety and damage to collateral structures. Sixteen studies met inclusion criteria. IRE was most commonly applied to the ventricular myocardium (n = 7/16, 44%) by a LifePak 9 Defibrillator (n = 9/16, 56%), NanoKnife Generator (n = 2/16, 13%), or other custom generators (n = 5/16, 31%). There was significant heterogeneity regarding electroporation protocols. On histological analysis, IRE was successful in creating ablation lesions with variable transmurality depending on the electric pulse parameters and catheter used. Preclinical studies suggest that cardiac tissue ablation using IRE shows promise in delivering efficacious, safe lesions.

PROPERTIES OF THE ALUMINUM-GALLIUM ARSENIDE IN THE PULSED ELECTRIC FIELD MODE

In the development of modern semiconductor devices are increasingly used multicomponent semiconductors. One of the well-proven ternary compounds of the materials of the group is aluminum-gallium arsenide AlxGa1-xAs. For AlxGa1-xAs the transport properties in high electric fields have been investigated. One of the problems is the insufficient knowledge of the transport properties of charge carriers in a pulsed field mode.The purpose of the paper is to study the drift processes during the pulsed mode of the electric field in aluminum-gallium arsenide AlxGa1-xAs (at x = 0.225).The modeling was carried out on the basis of the relaxation equations for the conservation of momentum, energy and concentration. Using the two-valley ГL-model, calculations were performed for each valley and averaged taking into account their population. The most typical electron scattering mechanisms have been researched: impurity (at neutral atoms and impurity ions) and phonon (acoustic, optical, and inter-valley). The temperature dependence of the pulse scattering rates was analyzed, and the electron mobility was calculated. It is shown that the result of the mobility simulation is in а good agreement with the experiment. The effect of the "overshoot" of the drift velocity is investigated. For Al0.225Ga0.775As, a numerical experiment was conducted on the effect of the electric field pulse parameters on the drift velocity: amplitudes of the field strength, pulse duration, and front magnitude.It is shown that the pulse-like change of the field leads to a short "overshoot" of the drift velocity. Greater field amplitude corresponds to an increase in the maximum value of the drift velocity. However, this is accompanied by a reduction in the time for which the effect of the "overshoot" appears. The pulse duration of the electric field does not affect the peak value of the drift velocity. The increase of the pulse front duration leads to a significant decrease in the transport properties in strong electric fields. A comparison is made of the peak values of the drift velocity and the length of the “ballistic range” of the electron in aluminum-gallium arsenide Al0.225Ga0.775As and gallium arsenide GaAs.

Using extracellular calcium concentration and electric pulse conditions to tune platelet-rich plasma growth factor release and clotting

Platelet-rich plasma (PRP) is an emerging autologous biologic method for wound healing. Clinicians apply PRP either topically (where it is activated ex-vivo before treatment by adding an external agent to trigger clotting and the release of growth factors that facilitate wound healing) or through injection (where it is activated in vivo at the injury site with no prior activation before injection). Because topical PRP activation typically utilizes bovine thrombin, which has significant potential side effects and high costs, recent studies have assessed the efficacy of combining extracellular calcium (EC) and electric pulses (EPs) to activate PRP. The potential to apply this novel technique to PRP both topically and internally via injection raises the question about the ability to tune the clotting time and growth factor release for a given application. While previous studies have assessed the impact of applying EPs of various durations either directly (conductive coupling) or indirectly (capacitive coupling) to PRP containing EC, no studies have assessed the tunability of this activation based on modifying EP parameters, EP delivery method (conductive or capacitive coupling), and the EC concentration. We hypothesize that tuning these parameters will modify intracellular calcium uptake to permit the control of growth factor release and clotting time, which are critical for optimizing PRP for either topical or internal clinical applications. A pilot study for a single donor demonstrates the potential for tunability as a function of the intensity of membrane manipulation and calcium concentration, which facilitate the increase of cytosolic calcium. This motivates future studies assessing EC and EP optimization and in vivo studies to determine the overall efficacy of this tunability for wound healing.

Electroporation-Induced Stress Response and Its Effect on Gene Electrotransfer Efficacy: In Vivo Imaging and Numerical Modeling.

Skin is an attractive target tissue for gene transfer due to its size, accessibility, and its immune competence. One of the promising delivery methods is gene delivery by means of electroporation (EP), i.e., gene electrotransfer (GET). To assess the importance of different effects of electroporation for successful GET we investigated: stress response and transfection efficacy upon different pulse protocols. Moreover, numerical modeling was used to explain experimental results and to test the agreement of experimental results with current knowledge about GET. Double transgenic mice Hspa1b-LucF (+/+) Hspa1b-mPlum (+/+) were used to determine the level of stress sensed by the cell in the tissue in vivo that was exposed to EP. The effect of five different pulse protocols on stress levels sensed by the exposed cells and their efficacy for gene electrotransfer for two plasmids pEGFP-C1 (EGFP) and pCMV-tdTomato was tested. Quantification of the bioluminescence signal intensity shows that EP, regardless of the electric pulse parameters used, increased mean bioluminescence compared to the baseline bioluminescence signal of the non-exposed skin. The results of numerical modeling indicate that thermal stress alone is not sufficient to explain the measured bioluminescence signal. Of the tested pulse protocols, the highest expression of EGFP and tdTomato was achieved with HV-MV (high voltage - medium voltage) protocols, which agrees also with numerical model. Although EP is widely used as a method for gene delivery, we show that the field could benefit from the use of mathematical modeling by introducing additional parameters such as EP induced stress and electrophoretic movement of plasmids.

Safe and efficient novel approach for non-invasive gene electrotransfer to skin

Gene transfer into cells or tissue by application of electric pulses (i.e. gene electrotransfer (GET)) is a non-viral gene delivery method that is becoming increasingly attractive for clinical applications. In order to make GET progress to wide clinical usage its efficacy needs to be improved and the safety of the method has to be confirmed. Therefore, the aim of our study was to increase GET efficacy in skin, by optimizing electric pulse parameters and the design of electrodes. We evaluated the safety of our novel approach by assaying the thermal stress effect of GET conditions and the biodistribution of a cytokine expressing plasmid. Transfection efficacy of different pulse parameters was determined using two reporter genes encoding for the green fluorescent protein (GFP) and the tdTomato fluorescent protein, respectively. GET was performed using non-invasive contact electrodes immediately after intradermal injection of plasmid DNA into mouse skin. Fluorescence imaging of transfected skin showed that a sophistication in the pulse parameters could be selected to get greater transfection efficacy in comparison to the standard ones. Delivery of electric pulses only mildly induced expression of the heat shock protein Hsp70 in a luminescent reporting transgenic mouse model, demonstrating that there were no drastic stress effects. The plasmid was not detected in other organs and was found only at the site of treatment for a limited period of time. In conclusion, we set up a novel approach for GET combining new electric field parameters with high voltage short pulses and medium voltage long pulses using contact electrodes, to obtain a high expression of both fluorescent reporter and therapeutic genes while showing full safety in living animals.

Structural Scale Levels of Plastic Deformation and Fracture of High-Strength Titanium Alloy Welds

Structural scale levels of plastic deformation and fracture of welded joints have been studied for two high-strength titanium alloys with a low (VT18U alloy) and a high (VT23 alloy) content of the bcc s phase. Ultrasonic forging and its combination with high-current pulsed electron beam treatment were used to activate nanoscale structural levels of deformation and fracture in welds in order to increase the fatigue life of welded structures. Ultrasonic forging provides an effective dispersion and nanostructuring of surface layers in the VT18U welded joints with a 4.6-fold increase in their fatigue life. The dispersion and nanostructuring of the VT23 laser welded joints is achieved only by ultrasonic forging combined with high-current electric pulse treatment, in which longitudinal dispersion of s bands occurs with the formation of orthorhombic a " nanolaths. In so doing, the fatigue life of the VT23 welds increases twice, but the effect depends on the power of the high-current generator and electrical pulse parameters. The fracture micrographs of the treated VT23 welded joints reveal nanofibrous bands responsible for ductile fracture and for the reduction of the fatigue crack growth rate. The structural changes and the increase in the fatigue life of the studied titanium alloy welds are associated with the activation of nanoscale structural levels of deformation and fracture induced by ultrasonic forging or by its combination with high-current pulsed electron beam treatment.

Numerical study of gene electrotransfer efficiency based on electroporation volume and electrophoretic movement of plasmid DNA

BackgroundThe efficiency of gene electrotransfer, an electroporation-based method for delivery of pDNA into target tissues, depends on several processes. The method relies on application of electric pulses with appropriate amplitude and pulse duration. A careful choice of electric pulse parameters is required to obtain the appropriate electric field distribution, which not only controls the electroporated volume, but also affects the movement of pDNA. We used numerical modeling to assess the influence of different types of electrodes and pulse parameters on reversibly electroporated volume and on the extent of pDNA–membrane interaction, which is necessary for successful gene electrotransfer.MethodsA 3D geometry was built representing the mice skin tissue and intradermally injected plasmid volume. The geometry of three different types of electrodes (plate, finger, needle) was built according to the configuration and placement of electrodes used in previously reported in vivo experiments of gene electrotransfer. Electric field distribution, resulting from different pulse protocols was determined, which served for calculation of reversible electroporation volume and for simulation of electrophoretic movement of pDNA. The efficiency of gene electrotransfer was evaluated in terms of predicted amount of pDNA present inside the volume of reversible electroporation at the end of pulse delivery.ResultsAccording to results of our numerical study, finger and needle electrodes provide larger amount of pDNA inside the volume of reversible electroporation than plate electrodes. However, these results are not consistent with the experiments showing that plate electrodes achieve the best transfection efficiency. Some inconsistencies were observed also by comparing the efficiencies of different high and low voltage pulse combinations, delivered by plate electrodes. The reason for inconsistencies probably lies in insufficient knowledge regarding the electroporation of stratum corneum. Namely, the size of the regions with high electrical conductivity, created by electroporation, was found to strongly affect predicted transfection efficiency.ConclusionsThe presented numerical model simulates the two most important processes involved in gene electrotransfer: electroporation of cells, and electrophoretic movement of pDNA. The inconsistencies between the model and experiments indicate incomplete knowledge of skin electroporation, or the involvement of other mechanisms, whose importance has not been yet identified.

Selective electrical stimulation of vagus nerve induces specific cytokine response.

Abstract Neuro-immune communication and neural circuits regulating immunity have been therapeutically explored in preclinical models and successfully in recent human clinical trials. Implantable bioelectronic devices that stimulate the inflammatory reflex are effective in suppressing cytokine production during endoxtoxemia and other models of infection and injury. Whether vagus nerve stimulation can enhance cytokines was previously unknown. Here we analyzed serum cytokine levels in response to electrical stimulation of the vagus nerve using different pulse parameters. The cervical vagus nerve was isolated in adult male Balb/C mice and subjected to asymmetric charge-balanced stimulation using bipolar cuff electrodes. Animals were stimulated for 4 min using the following parameters: frequency 30–200 Hz, amplitude 50–750 μA, pulse width 50–250 μs. After 2 hrs, blood was collected and serum levels of TNFα, IL-6, IL-10, IL-12, and KC/GRO determined. Serum cytokine levels changed significantly (p&amp;lt;0.0001–0.05) and exhibited tuning curve properties when subjected to varying electrical pulse parameters, suggesting selective recruitment of efferent or afferent fibers that regulate specific cytokine production. Further insight into the nature of the neural circuits was ascertained through unilateral and bilateral vagotomies, distal and proximal to the stimulation location. Together, these studies demonstrate that systemic cytokine levels can be modulated by selectively stimulating the vagus nerve using specific combinations of frequency, amplitude, and pulse width. This study was funded in part by DARPA (HR0011-15-2-0016) and NIH (1R35GM118182-01).

Comprehensive Characterization of the Interaction between Pulsed Electric Fields and Live Cells by Confocal Raman Microspectroscopy.

This study reports a comprehensive analysis of the effect of 100 μs electric pulses on the biochemical composition of live cells using a label-free approach, confocal Raman microspectroscopy. We investigated different regions of interest around the nucleus of the cells and the dose-effect relationship related to different electric pulse parameters. We also extended the study to another cell type. Membrane resealing was monitored by pulsing the cells in reversible or irreversible electropermeabilization condition at different temperatures. Our results confirmed a previous publication showing that proteins and lipids were highly impacted by the delivery of electric pulses. These chemical changes were similar in different locations around the cell nucleus. By sweeping the field magnitude, the number of electric pulses, or their repetition rate, the Raman signatures of live cells appeared to be related to the electropermeabilization state, verified by Yo-Pro-1 uptake. We also demonstrated that the chemical changes in the Raman signatures were cell-dependent even if common features were noticed between the two cell types used.

Deagglomeration of nanostructured aluminum oxyhydroxide upon shock wave impact of electrohydraulic discharge

This article discusses the patterns of deagglomeration of aluminum oxyhydroxide nanosheets upon electrohydraulic discharge. The dispersion efficiency by electrohydraulic discharge is higher than that of ultrasound impact. The highest influence on destruction efficiency is exerted by the time of energy release, which determines the intensity of cavitation processes in water. No disintegration of initial nanostructures is observed under the selected parameters of electric pulses.

Use of electroacupuncture and transcutaneous electrical acupoint stimulation in reproductive medicine: a group consensus.

With the rapid development of assisted reproductive technology, various reproductive disorders have been effectively addressed. Acupuncture-like therapies, including electroacupuncture (EA) and transcutaneous electrical acupoint stimulation (TEAS), become more popular world-wide. Increasing evidence has demonstrated that EA and TEAS are effective in treating gynecological disorders, especially infertility. This present paper describes how to select acupoints for the treatment of infertility from the view of theories of traditional Chinese medicine and how to determine critical parameters of electric pulses of EA/TEAS based on results from animal and clinical studies. It summarizes the principles of clinical application of EA/TEAS in treating various kinds of reproductive disorders, such as polycystic ovary syndrome (PCOS), pain induced by oocyte retrieval, diminished ovarian reserve, embryo transfer, and oligospermia/ asthenospermia. The possible underlying mechanisms mediating the therapeutic effects of EA/TEAS in reproductive medicine are also examined.

Caspase dependent apoptosis induced in yeast cells by nanosecond pulsed electric fields

Saccharomyces cerevisiae yeast cells were used as a model organism to investigate the effects of various pulsed electric fields on the programed death of such cells. These were exposed to electric field pulses with field strengths (E) of up to 220kV/cm. The effects of square shaped pulses having different durations (τ=10–90ns) and different pulse numbers (pn=1–5) were then analysed. The obtained results show that nanosecond pulses can induce the death of such cells, which in turn is dependent on the electric field pulse parameters and increase with the rise in E, τ and pn. The decrease of the cells' viability was accompanied by an increase in the active form of intracellular yeast metacaspases. It was thus shown that nanosecond electric field pulses induced the caspase-dependent yeast cell death.

Electrodepositing Ni-TiN nanocomposite Layers with Applying Action of Ultrasonic Waves

Ultrasonic wave was applied to prepare nanocermet Ni–TiN composite layers on steel substrates during electrodeposition. The action of mechanical disturbance by ultrasonic waves on electrolyte mass transfer, the inhibition of nanoparticle aggregation by ultrasonic cavitation and the effect of electric pulse parameters on the nucleation and growth of grains were investigated. Proper utilization of the mechanical disturbance and cavitation effects of ultrasonic waves could improve the mass transfer of the electrolyte and inhibit the aggregation of TiN nanoparticles, leading to homogeneous dispersion of the particles in the plated layer. Electricity pulses of high amplitude, narrow width and low occupancy accelerated the nucleation and inhibited the growth of metal grains, resulting in nano-sized nickel grains of homogeneous size. The nanocermet Ni–TiN composite layer consisted of nanocrystalline nickel (40–60 nm) and TiN nanoparticles was obtained on the surface of common metal substrates using optimum conditions.

Electric field enhanced 3D scalable low-voltage nano-spike electroporation system

Electroporation (EP) is one of the most widely used methods for the introduction of bio-molecules into the cells due to its high efficiencies, simplicity, and safety. Previous macro- and micro-scale EP systems suffer from drawbacks such as costly and time-consuming fabrication processes, high voltage operation causes undesirable electrochemical reactions, low cell viabilities, and electrode degradation. In this paper, we presented a low-cost three-dimensional (3D) scalable nano-spike electroporation system for efficient molecules delivery with high cell viabilities at low applied voltages. Arrays of 3D Aluminum (Al) nano-spikes (NSPs) were fabricated through scalable, reproducible and cost effective electrochemical anodization and etching processes. Due to scalability of the fabrication process, 3D NSPs were fabricated on chips as well as at the wafer level for large scale processing. 3D nano-spike electroporation (NSP-EP) chips were capable of handling small cell populations (100–500) while NSP-EP wafers can handle large cell populations (104–105). Electroporation at low voltages is obtained due to electric field enhancement at high-aspect-ratio NSPs. With same electric field strength, high EP efficiencies ƞEP and cell viability фcell (>93±6%) were obtained at more than ten times lower voltages (2V) on NSP-EP chips as compared to planar electroporation (PEP) devices without NSPs. By optimizing electric pulse parameters and nano-spikes dimensions, NSP-EP chip performance was enhanced by minimizing undesirable electrochemical reactions and electrolysis that were observed on PEP devices due to high voltage operations.

Photoexcitation of electron wave packets in quantum spin Hall edge states: Effects of chiral anomaly from a localized electric pulse

We show that, when a spatially localised electric pulse is applied at the edge of a quantum spin Hall system, electron wavepackets of the helical states can be photoexcited by purely intra-branch electrical transitions, without invoking the bulk states or the magnetic Zeeman coupling. In particular, as long as the electric pulse remains applied, the photoexcited densities lose their character of right- and left-movers, whereas after the ending of the pulse they propagate in opposite directions without dispersion, i.e. maintaining their space profile unaltered. Notably we find that, while the momentum distribution of the photoexcited wave packets depends on the temperature $T$ and the chemical potential $\mu$ of the initial equilibrium state and displays a non-linear behavior on the amplitude of the applied pulse, in the mesoscopic regime the space profile of the wave packets is independent of $T$ and $\mu$. Instead, it depends purely on the applied electric pulse, in a linear manner, as a signature of the chiral anomaly characterising massless Dirac electrons. We also discuss how the photoexcited wave packets can be tailored with the electric pulse parameters, for both low and finite frequencies.

Effects of duration of electric pulse on in vitro development of cloned cat embryos with human artificial chromosome vector.

The current applications for cat cloning include production of models for the study of human and animal diseases. This study was conducted to investigate the optimal fusion protocol on in vitro development of transgenic cloned cat embryos by comparing duration of electric pulse. Cat fibroblast cells containing a human artificial chromosome (HAC) vector were used as genetically modified nuclear donor cells. Couplets were fused and activated simultaneously with a single DC pulse of 3.0kV/cm for either 30 or 60μs. Low rates of fusion and embryo development to the blastocyst stage were observed in the reconstructed HAC-transchromosomic embryos, when the duration of fusion was prolonged to 60μs. In contrast, the prolongation of electric pulse duration improved the embryo development and quality in the reconstructed control embryos without HAC vector. Our results suggested that the optimal parameters of electric pulses for fusion in cat somatic cell nuclear transfer vary among the types used for donor cells.