Electroporation Procedure Research Articles

Threshold Interphase Delay for Bipolar Pulses to Prevent Cancellation Phenomenon during Electrochemotherapy.

Electroporation-based procedures employing nanosecond bipolar pulses are commonly linked to an undesirable phenomenon known as the cancelation effect. The cancellation effect arises when the second pulse partially or completely neutralizes the effects of the first pulse, simultaneously diminishing cells' plasma membrane permeabilization and the overall efficiency of the procedure. Introducing a temporal gap between the positive and negative phases of the bipolar pulses during electroporation procedures may help to overcome the cancellation phenomenon; however, the exact thresholds are not yet known. Therefore, in this work, we have tested the influence of different interphase delay values (from 0 ms to 95 ms) using symmetric bipolar nanoseconds (300 and 500 ns) on cell permeabilization using 10 Hz, 100 Hz, and 1 kHz protocols. As a model mouse hepatoma, the MH-22a cell line was employed. Additionally, we conducted in vitro electrochemotherapy with cisplatin, employing reduced interphase delay values (0 ms and 0.1 ms) at 10 Hz. Cell plasma membrane permeabilization and viability dependence on a variety of bipolar pulsed electric field protocols were characterized. It was shown that it is possible to minimize bipolar cancellation, enabling treatment efficiency comparable to monophasic pulses with identical parameters. At the same time, it was highlighted that bipolar cancellation has a significant influence on permeabilization, while the effects on the outcome of electrochemotherapy are minimal.

Optimisation of DNA electroporation protocols for different plant-associated bacteria

Electroporation is a vital process that facilitates the use of modern recombineering and other high-throughput techniques in a wide array of microorganisms, including non-model bacteria like plant growth-promoting bacteria (PGPB). These microorganisms play a significant role in plant health by colonizing plants and promoting growth through nutrient exchange and hormonal regulation. In this study, we introduce a sequential Design of Experiments (DOE) approach to obtain highly competent cells swiftly and reliably for electroporation. Our method focuses on optimizing the three stages of the electroporation procedure—preparing competent cells, applying the electric pulse field, and recovering transformed cells—separately. We utilized a split-plot fractional design with five factors and a covariate to optimize the first step, response surface methodology (RSM) for the second step, and Plackett-Burman design for two categorical factors and one continuous factor for the final step. Following the experimental sequence with three bacterial models, we achieved efficiencies 10 to 100 times higher, reaching orders of 105 to 106 CFU/μg of circular plasmid DNA. These results highlight the significant potential for enhancing electroporation protocols for non-model bacteria.

Engineering probiotic bacteria to express tcdB antigen as an oral vaccine carrier against Clostridium difficile infection

Background Clostridium (now known as Clostridioides) difficile (C. difficile) is a spore-forming, gram-positive organism that can pose serious public health complications. The elderly are especially vulnerable to C. difficile infections, which can be fatal. C. difficile strains cause symptomatic diseases via the release of two toxins; tcdA and tcdB, that induce inflammation and tissue damage. Vaccines targeting any of these toxins may offer an effective strategy against C. difficile infections. Objective This study aimed to use live probiotics as oral vaccines to express the C. difficile toxin B gene. Oral vaccination has many advantages over intramuscular injection, as it has higher compliance, feasibility, and simpler administration. In addition, oral vaccines can stimulate both mucosal and systemic immunity against the target antigen. Probiotic bacteria were chosen as they present ideal candidates for this goal in terms of safety and health promotion. Materials and methods We chose two probiotic strains: Lactobacillus gasseri ATCC 33323 in addition to Enterococcus faecium NM1015, which had previously been identified in our lab and is capable of suppressing C. difficile colonization. The C-terminal of the tcdB gene was amplified by polymerase chain reaction (PCR) from C. difficile, cloned, and transformed into an E. coli EZ strain. The tcdB fragment was digested with BamHI and XhoI (NEB, UK) and subcloned into the bile salt-inducible expression plasmid pLB210 (obtained from INRA, France). The transformation and electroporation procedures were employed to insert cloning and expression plasmids into the target bacteria. Colony PCR was used to confirm the engineering strains. Reverse transcription PCR was used to confirm the expression of the C. difficile tcdB fragment. Results and conclusion The expression vector ‘p210-tcdB’ was constructed, then introduced into the selected probiotic strains by electroporation and confirmed by colony PCR and plasmid extraction. The reverse transcription (RT)-PCR confirmed the expression of the gene by the engineered strains. No significant difference in the survival rate was observed between the engineered strains and their parental types at pH 2.00 and 1% oxygen. Moreover, the strains showed satisfactory plasmid stability for 210 generations. Future work will involve the in vivo evaluation of the engineered probiotic strains as oral vaccines against C. difficile using an animal model.

Treatment of Spontaneous Tumors With Algorithmically Controlled Electroporation.

To study the safety and efficacy of algorithmically controlled electroporation (ACE) against spontaneous equine melanoma. A custom temperature sensing coaxial electrode was paired with a high voltage pulse generation system with integrated temperature feedback controls. Computational modeling and ex vivo studies were conducted to evaluate the system's ability to achieve and maintain target temperatures. Twenty-five equine melanoma tumors were treated with a 2000 V protocol consisting of a 2-5-2 waveform, 45 °C temperature set point, and integrated energized times of 0.005 s, 0.01 s, or 0.02 s (2500x, 5000x, and 10000x 2 μs pulses, respectively). Patients returned 20-50 days post treatment to determine the efficacy of the treatment. ACE temperature control algorithms successfully achieved and maintained target temperatures in a diverse population of spontaneous tumors with significant variation in tissue impedance. All treatments were completed successfully without and without adverse events. Complete response rates greater than 93% were achieved in all treatment groups. ACE is a safe and effective treatment for spontaneous equine melanoma. The temperature control algorithm enabled rapid delivery of electroporation treatments without prior knowledge of tissue electrical or thermal properties and could adjust to real time changes in tissue properties. Real time temperature control in electroporation procedures enables treatments near critical structures where thermal damage is contraindicated. Unlike standard approaches, ACE protocols do not require extensive pretreatment planning or knowledge of tissue properties to determine an optimal energy delivery rate and they can account for changes in tissue state (e.g., perfusion) in real time to simultaneously minimize treatment time and potential for thermal damage.

A Novel Strategy to Produce CAR-Γδ T Cells Via Targeted Knockout By CRISPR/Cas9 and Targeted Knockin By AAV

Chimeric antigen receptor (CAR)-γδ T cells are promising in immunotherapy owing to the dual recognition system of γδ T cells, which is potential to exhibit stronger anti-tumor effect. Up to date, established CAR-γδ T cells exhibit poor anti-tumor efficiency and are commonly generated using lentiviral (LV) or gamma-retroviral (γRV) vectors, which may increase the risk of insertional mutagenesis. Nowadays, clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) has been widely used to cleave the genomic sequence precisely, as well as Adeno-associated Virus (AAV) being used to deliver DNA templates for target sequence knock-in (KI) mediated by homology-directed repair, which could contribute to achieve the accurate insertion of CAR at a specific site. Furthermore, the endogenous promoter of TCR has been used to express CAR molecules with stable expression and better antitumor function in the production of CAR-αβ T cells. In the present study, we established and optimized the strategy to generate CAR-γδ T cells, combining the site-specific knock-out (KO) targeting the TCR delta chain constant region (TRDC) site of γδ T cells using CRISPR/Cas9 and site-specific CAR knock-in using AAV. Firstly, we screened out a functional guide RNA (gRNA) targeting the γδ TCR TRDC locus by prediction tools and validation in cell line model experiments, and achieved an INDEL frequency of 31.8%, which showed a high activity of gRNA. Then, we optimized the option to increase KO efficiency including exploring appropriate Cas9 amount, ratio of gRNA to Cas9 and cell number titration. Besides, we designed AAV vector carrying CAR sequence targeting the gRNA aiming site selected above (Figure1A). After that, we used CRISPR/Cas9 and AAV to knock out TCR and knock in CAR sequence in γδ T cells. We tried different electroporation procedures (X001, T007, U014, T023) and cell culture conditions (cultured in medium containing zoledronic acid (ZOL)/ IL2 for 5 days or for 10 days) to select out the optimal procedure to acquire high KO/KI efficiency and high cell proliferation rate. When we used PBMC being cultured in medium containing zoledronic acid (ZOL)/ IL2 for 5 days (Day 5) to generate CAR-γδ T cells, cells electroporated using X001 procedure showed the best TCR KO efficiency of ~80%（p<0.05）and CAR expression of ~20% (p< 0.01), as well as keeping the highest cell proliferation rate than other procedures (p<0.0001) (Figure 1B and 1C). If we used PBMC being cultured in medium containing zoledronic acid (ZOL)/ IL2 for 10 days (Day 10) to generate CAR-γδ T cells, U014 program also exhibited a relative high CAR expression and the significant quicker cell growth than others in the later period (p<0.01) (Figure 1B and 1C). However, the later proliferation rate of CAR-γδ T cells generated at Day 5 was superior to that generated at Day10. Thus, we determined that a 5 days' cell culture and the use of X001 program were the optimal condition to generate CAR-γδ T cells. Besides, we found that KI efficiency is positively correlative with Multiplicity of infection (MOI). While MOI comes to 1.2×10 5, the KI efficiency couldn't be increased (Figure 1D). Finally, we evaluated the anti-tumor function of CAR-γδ T cells . Antigen stimulated CAR-γδ T cells significantly released TNFα and INFγ, but failed to produce IL2 (Figure 1E). Besides, CAR-γδ T cells could significantly lysis different tumor cells even at a low effector/target ratio (E/T) (Figure 1G). Interestingly, the proportion of memory phenotype (CD62L + ) in CAR-γδ T cells after being stimulated was comparable to that of CAR-γδ T cells without stimulation, indicating the potential of the great anti-tumor activity of CAR-γδ T cells after being stimulated in tumor environment (Figure 1F). Our new method successfully generated CAR-γδ T cells with a CAR expression of 20% and in a high proliferation rate, which was determined to exert effective anti-tumor effects.

Bioactivated and PEG-Protected Circa 2nm Gold Nanoparticles for in Cell Labelling and Cryo-Electron Microscopy.

Advances in cryo-electron microscopy (EM) enable imaging of protein assemblies within mammalian cells in a near native state when samples are preserved by cryogenic vitrification. To accompany this progress, specialized EM labelling protocols must be developed. Gold nanoparticles (AuNPs) of 2nm are synthesized and functionalized to bind selected intracellular targets inside living human cells and to be detected in vitreous sections. As a proof of concept, thioaminobenzoate-, thionitrobenzoate-coordinated gold nanoparticles are functionalized on their surface with SV40 Nuclear Localization Signal (NLS)-containing peptides and 2kDa polyethyleneglycols (PEG) by thiolate exchange to target the importin-mediated nuclear machinery and facilitate cytosolic diffusion by shielding the AuNP surface from non-specific binding to cell components, respectively. After delivery by electroporation into the cytoplasm of living human cells, the PEG-coated AuNPs diffuse freely in the cytoplasm but do not enter the nucleus. Incorporation of NLS within the PEG coverage promotes a quick nuclear import of the nanoparticles in relation to the density of NLS onto the AuNPs. Cryo-EM of vitreous cell sections demonstrate the presence of 2nm AuNPs as single entities in the nucleus. Biofunctionalized AuNPs combined with live-cell electroporation procedures are thus potent labeling tools for the identification of macromolecules in cellular cryo-EM.

Development of high-copy number plasmids in Pseudoalteromonas haloplanktis TAC125

The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) is considered an interesting alternative host for the recombinant protein production, that can be explored when the conventional bacterial expression systems fail. Indeed, the manufacture of all the difficult-to-express proteins produced so far in this bacterial platform gave back soluble and active products. Despite these promising results, the low yield of recombinant protein production achieved is hampering the wider and industrial exploitation of this psychrophilic cell factory. All the expression plasmids developed so far in PhTAC125 are based on the origin of replication of the endogenous pMtBL plasmid and are maintained at a very low copy number. In this work, we set up an experimental strategy to select mutated OriR sequences endowed with the ability to establish recombinant plasmids at higher multiplicity per cell. The solution to this major production bottleneck was achieved by the construction of a library of psychrophilic vectors, each containing a randomly mutated version of pMtBL OriR, and its screening by fluorescence-activated cell sorting (FACS). The selected clones allowed the identification of mutated OriR sequences effective in enhancing the plasmid copy number of approximately two orders of magnitude, and the production of the recombinant green fluorescent protein was increased up to twenty times approximately. Moreover, the molecular characterization of the different mutant OriR sequences allowed us to suggest some preliminary clues on the pMtBL replication mechanism that deserve to be further investigated in the future.Key points• Setup of an electroporation procedure for Pseudoalteromonas haloplanktis TAC125.• Two order of magnitude improvement of OriR-derived psychrophilic expression systems.• Almost twenty times enhancement in Green fluorescent protein production.

Predicting the chemosensitivity of pancreatic cancer cells as a personalized therapy.

Annually, approx. 4000 patients are diagnosed with pancreatic cancer in Poland, and the number of deaths is close to the number of diagnoses. Such a high morbidity/mortality ratio is caused by a high percentage of unresectable lesions (about 80%) and chemoresistance, which, among other things, is due to the specific desmoplastic environment. Currently, there are 2 main systemic treatment regimens for pancreatic cancer: FOLFIRINOX (which is a combination of folic acid, fluorouracil (5-FU), irinotecan, and oxaliplatin) and combined treatment with nab-paclitaxel plus gemcitabine (NPXL+GMC). In order to increase the effectiveness of systemic treatments for individual patients, cell lines derived from resected pancreatic tumors were developed and their chemosensitivity to various agents was examined. The hypothesis was that patients may benefit from individualization of chemotherapy. Patients with histopathologically confirmed pancreatic cancer were operated on using irreversible electroporation (IRE) procedure. After isolating and establishing individual cell lines, chemosensitivity to 5-FU, GMC and NPXL was determined using MTT assay in primary and metastatic cell cultures. Three primary cell lines were isolated for the prediction of chemosensitivity. Gemcitabine was shown to be more effective at lower doses compared to 5-FU, while NPXL was more effective than 5-FU, and both of these were less effective in metastatic cells. Pancreatic cancer cell chemoresistance was confirmed in stage IV. Determination of chemosensitivity profiles using cell lines may help in the selection of systemic treatments for individual patients. This method can be the basis for a personalized planned chemotherapeutic protocol.

Toolbox for Genetic Transformation of Non-Conventional Saccharomycotina Yeasts: High Efficiency Transformation of Yeasts Belonging to the Schwanniomyces Genus.

Non-conventional yeasts are increasingly being investigated and used as producers in biotechnological processes which often offer advantages in comparison to traditional and well-established systems. Most biotechnologically interesting non-conventional yeasts belong to the Saccharomycotina subphylum, including those already in use (Pichia pastoris, Yarrowia lypolitica, etc.), as well as those that are promising but as yet insufficiently characterized. Moreover, for many of these yeasts the basic tools of genetic engineering needed for strain construction, including a procedure for efficient genetic transformation, heterologous protein expression and precise genetic modification, are lacking. The first aim of this study was to construct a set of integrative and replicative plasmids which can be used in various yeasts across the Saccharomycotina subphylum. Additionally, we demonstrate here that the electroporation procedure we developed earlier for transformation of B. bruxellensis can be applied in various yeasts which, together with the constructed plasmids, makes a solid starting point when approaching a transformation of yeasts form the Saccharomycotina subphylum. To provide a proof of principle, we successfully transformed three species from the Schwanniomyces genus (S. polymorphus var. polymorphus, S. polymorphus var. africanus and S. pseudopolymorphus) with high efficiencies (up to 8 × 103 in case of illegitimate integration of non-homologous linear DNA and up to 4.7 × 105 in case of replicative plasmid). For the latter two species this is the first reported genetic transformation. Moreover, we found that a plasmid carrying replication origin from Scheffersomyces stipitis can be used as a replicative plasmid for these three Schwanniomyces species.

Introduction of loxP sites by electroporation in the mouse genome; a simple approach for conditional allele generation in complex targeting loci

BackgroundThe discovery of the CRISPR-Cas9 system and its applicability in mammalian embryos has revolutionized the way we generate genetically engineered animal models. To date, models harbouring conditional alleles (i.e. two loxP sites flanking an exon or a critical DNA sequence of interest) are amongst the most widely requested project type that are challenging to generate as they require simultaneous cleavage of the genome using two guides in order to properly integrate the repair template. An approach, using embryo sequential electroporation has been reported in the literature to successfully introduce loxP sites on the same allele. Here, we describe a modification of this sequential electroporation procedure that demonstrated the production of conditional allele mouse models for eight different genes via one of two possible strategies: either by consecutive sequential electroporation (strategy A) or non-consecutive sequential electroporation (strategy B). This latest strategy originated from using the by-product produced when using consecutive sequential electroporation (i.e. mice with a single targeted loxP site) to complete the project.ResultsBy using strategy A, we demonstrated successful generation of conditional allele models for three different genes (Icam1, Lox, and Sar1b), with targeting efficiencies varying between 5 and 13%. By using strategy B, we generated five conditional allele models (Loxl1, Pard6a, Pard6g, Clcf1, and Mapkapk5), with targeting efficiencies varying between 3 and 25%.ConclusionOur modified electroporation-based approach, involving one of the two alternative strategies, allowed the production of conditional allele models for eight different genes via two different possible paths. This reproducible method will serve as another reliable approach in addition to other well-established methodologies in the literature for conditional allele mouse model generation.

An Optimized Electroporation Procedure for Genetic Transformation of Candida auris.

Candida auris is responsible for recent outbreaks with significant mortality in hospitalized and long-term care patients. As a highly transmissible and multidrug-resistant fungal pathogen, genetic tools are urgently needed for deciphering mechanisms involved in the host-pathogen interactions and potentially identifying new fungal targets for therapeutic development. Here, we provide a reliable transformation protocol based on an efficient electroporation procedure and the use of a mycophenolic acid resistance marker.

Optimized Protocol for Plasma-Derived Extracellular Vesicles Loading with Synthetic miRNA Mimic Using Electroporation.

Extracellular vesicles (EVs) are a population of particles naturally released by cells to transport biological messages, including nucleic acids. Thus, EVs represent an ideal vehicle to deliver therapeutic miRNAs. The current challenge is the development of efficient protocols to load EVs with exogenous miRNAs. Human plasma is an abundant source of EVs which can be manipulated for therapeutic applications. Despite numerous techniques are currently available to load EVs, all of them present issues which limit their clinical application. Among all, electroporation was shown to be superior to other protocols and to efficiently load plasma-derived EVs with miRNAs. However, also the electroporation procedure presents issues that can reduce the miRNA delivery. In this chapter, we describe a protocol to isolate EVs from human plasma, to load synthetic miRNA mimics using electroporation, to evaluate EV integrity and miRNA loading into EVs. Finally, the analysis of EV functionality allows to investigate the ability of engineered EVs to transfer the miRNAs to target cells and to exploit their biological effects.

An Electrochemical Protocol for CRISPR-Mediated Gene-Editing of Sheep Embryonic Fibroblast Cells

Genetic engineering of farm animals is commonly carried out via cell-mediated transfection followed by somatic cell nuclear transfer. However, efficient transfer of exogenous DNA into ovine embryonic fibroblast (EF) cells without compromising cell viability has remained a challenging issue. Here, we aimed to develop a protocol for electrotransfection of sheep EF cells. First, we optimized the pulsing condition using an OptiMEM-GlutaMAX medium as the electroporation buffer and found 2 pulses of 270 V, each for 10 ms and 10 s interval, is the most efficient condition to have a high rate of transfection and cell survival. Moreover, supplementing 3% dimethyl sulfoxide (DMSO) into the electroporation medium considerably improved the cell viability after the electroporation process. The electroporation procedure resulted in >98% transfection efficiency and >97% cell survival rate using reporter plasmids. Finally, using CRISPR/Cas9-encoding vectors, we targeted BMP15 and GDF9 genes in sheep EF cells. The electroporated cells are associated with a 52% indels rate using single gRNAs as well as a highly efficient target deletion using 2 gRNAs. In conclusion, we have developed an electrotransfection protocol using the OptiMEM-GlutaMAX medium supplemented with 3% DMSO for sheep EF cells. The electroporation method can be used for cell-mediated gene-editing in sheep.

TUB and ZNF532 Promote the Atoh1-Mediated Hair Cell Regeneration in Mouse Cochleae.

Hair cell (HC) regeneration is a promising therapy for permanent sensorineural hearing loss caused by HC loss in mammals. Atoh1 has been shown to convert supporting cells (SCs) to HCs in neonatal cochleae; its combinations with other factors can improve the efficiency of HC regeneration. To identify additional transcription factors for efficient Atoh1-mediated HC regeneration, here we optimized the electroporation procedure for explant culture of neonatal mouse organs of Corti and tested multiple transcription factors, Six2, Ikzf2, Lbh, Arid3b, Hmg20 a, Tub, Sall1, and Znf532, for their potential to promote Atoh1-mediated conversion of SCs to HCs. These transcription factors are expressed highly in HCs but differentially compared to the converted HCs based on previous studies, and are also potential co-reprograming factors for Atoh1-mediated SC-to-HC conversion by literature review. P0.5 cochlear explants were electroporated with these transcription factors alone or jointly with Atoh1. We found that Sox2+ progenitors concentrated within the lateral greater epithelial ridge (GER) can be electroporated efficiently with minimal HC damage. Atoh1 ectopic expression promoted HC regeneration in Sox2+ lateral GER cells. Transcription factors Tub and Znf532, but not the other six tested, promoted the HC regeneration mediated by Atoh1, consistent with previous studies that Isl1 promotes Atoh1-mediated HC conversionex vivo and in vivo and that both Tub and Znf532 are downstream targets of Isl1. Thus, our studies revealed an optimized electroporation method that can transfect the Sox2+ lateral GER cells efficiently with minimal damage to the endogenous HCs. Our results also demonstrate the importance of the Isl1/Tub/Znf532 pathway in promoting Atoh1-mediated HC regeneration.

No needle mesotherapy device in treatment of corticosteroid induced striae in systemic lupus erythematosus patients.

Introduction: is a needle free mesotheraputic device that is able to insert analytical pure active substance to the deep layers of skin within cell levels using the most modern electroporation procedure. Objectives: this study aimed to assess electroporation as a method of transdermal administration of medications in treatment of corticosteroid induced striae in systemic lupus erythematosus patients. Methods: Twenty female Systemic lupus erythematous (SLE) patients suffering from corticosteroid induced striae in different body parts, were included in this study. Patients were subjected to full history taking, complete clinical examination, and assessment of disease activity using systemic lupus erythematosus disease activity index (SLEDAI) score. Each patient received treatment in the form of six weekly successive cessions using electroporation device Mesostyle and photos were obtained after each cession. Results: We found marked improvement of striae appearance with noticeable patients’ satisfaction without any adverse effects. Conclusion: This study supports the role of electroporation which is safe, tolerable, non-invasive, and effective method for transdermal administration of medications.

Finite Element Analysis of Cell Killing Probability in Electroporation with Single Bipolar Electrode

Purpose: In the electroporation we can use different electrode types such as needle and plate electrode with different arrangements. One of the new electrode types is single bipolar electrode that the anode and cathode components are in the same needle for decreasing the invasiveness of electroporation procedure. Materials and Methods: For treatment planning purposes we can use different cell killing probability models such as Peleg-Fermi model. The aim of this study is to investigate the impact of geometric electrode parameters such as conductive pole length, insulated pole length and pulse voltage in bipolar electrode on the cell killing probability distribution in electroporation by COMSOL Multiphysics. Results: The target tissue volume with cell killing probability of >80% was increased with conductive pole length, and voltage and decreased with insulated pole length. Conclusion: This paper has highlighted the importance of conductive and insulated pole length and voltage in bipolar electrode on the cell killing probability distribution and electroporated volume in the EP.

Easily applicable modifications to electroporation conditions improve the transformation efficiency rates for rough morphotypes of fast-growing mycobacteria

Electroporation is the most widely used and efficient method to transform mycobacteria. Through this technique, fast- and slow-growing mycobacteria with smooth and rough morphotypes have been successfully transformed. However, transformation efficiencies differ widely between species and strains. In this study, the smooth and rough morphotypes of Mycobacteroides abscessus and Mycolicibacterium brumae were used to improve current electroporation procedures for fast-growing rough mycobacteria. The focus was on minimizing three well-known and challenging limitations: the mycobacterial restriction-modification systems, which degrade foreign DNA; clump formation of electrocompetent cells before electroporation; and electrical discharges during pulse delivery, which were reduced by using salt-free DNA solution. Herein, different strategies are presented that successfully address these three limitations and clearly improve the electroporation efficiencies over the current procedures. The results demonstrated that combining the developed strategies during electroporation is highly recommended for the transformation of fast-growing rough mycobacteria.

Multiphysics analysis of nsPEF induced electrodeformation in a dispersive cell model

Exposed to the nanosecond pulsed electric field (nsPEF), biological cells can be stretched in the direction parallel to the electric field direction. A multiphysics model to investigate electrodeformation of a spherical cell with double-layered plasma membrane accounting for both electroporation and dielectric relaxation of the membrane is proposed. Transmembrane potential, Maxwell stress tensor, total elastic strain energy, and deformation degree, the typical influential factors and indicators for electroporation and electrodeformation, are probed via the above multiphysics model under the action of unipolar and bipolar nsPEFs. The results suggest that the double-layered model can reflect the experimental cellular deformation more accurately than the single-layered model in that the long axis of the ellipsoid is stretched several micrometers in the double-layered model, while it is stretched several nanometers in the single-layered model. And merging the effect of dielectric relaxation into the model leads to a relatively lighter but faster deformation extent, and applying bipolar nsPEF alleviates the stretch for electrodeformation quantified with the lower aspect ratio of two principal radii of the ellipsoidal cell and the lower elastic strain energy. Our model can reflect the temporal evolution of electroporation and electrodeformation procedure more accurately, which is instructive to exert the nsPEF in biochemical experiments and clinical applications.

High-Efficiency Electroporation for Genetic Improvement of Fungal Strains.

Electroporation is a method for the introduction of molecules (usually nucleic acids) into a cell, consisting of submitting the cells to high-voltage and short electric pulses in the presence of the exogenous DNA/molecule. It is a versatile method, adaptable to different types of cells, from bacteria to cultured cells to higher eukaryotes, and thus has applications in many diverse fields, such as environmental biology, biotechnology, genetic engineering, and medicine. Electroporation has some advantages over other genetic transformation strategies, including the simplicity of the method, a wide range of adjustable parameters (possibility of optimization), high reproducibility and avoidance of the use of chemicals toxic to cells. Here we describe an optimized electroporation procedure for the industrially important fungus Acremonium chrysogenum, using germinated conidia and fragmented young mycelium. In both cases, the transformation efficiency was higher compared to the conventional polyethylene glycol (PEG)-mediated transformation of protoplasts.

Sub-microsecond electrotransfection using new modality of high frequency electroporation

Micro-millisecond range electric field pulses have been used for decades to facilitate DNA transfer into cells and tissues, while the growing number of clinical trials underline the strong potential of DNA electroporation. In this work, we present new sub-microsecond range protocols and methodology enabling successful electrotransfection in the sub-microsecond range. To facilitate DNA transfer, a 3 kV/60 A and high frequency (1 MHz) sub-microsecond range square wave generator was applied in the study. As a model, Chinese hamster ovary (CHO-K1) cells were used. Sub-microsecond range (300–700 ns) high frequency pulsed electric fields of 2–15 kV/cm were applied. The efficiency of electrotransfection was evaluated using two green fluorescent protein encoding plasmids of different size (3.5 kbp and 4.7 kbp). It was shown that transfection efficiency cannot be effectively improved with increase of the number of pulses after a certain threshold, however, independently on the plasmid size, the proposed sub-microsecond range pulsing methodology (2–5 kV/cm; n = 250) efficiency-wise was equivalent to 1.5 kV/cm × 100 μs × 4 electroporation procedure. The results of the study are useful for further development of in vitro and in vivo methods for effective electrotransfer of DNA using shorter pulses.