Electroporation Method Research Articles

Adipose tissue: a new target for electroporation-enhanced DNA vaccines

DNA vaccines delivered using electroporation (EP) have had clinical success, but these EP methods generally utilize invasive needle electrodes. Here, we demonstrate the delivery and immunogenicity of a DNA vaccine into subcutaneous adipose tissue cells using noninvasive EP. Using finite element analysis, we predicted that plate electrodes, when oriented properly, could effectively concentrate the electric field within adipose tissue. In practice, these electrodes generated widespread gene expression persisting for at least 60 days in vivo within interscapular subcutaneous fat pads of guinea pigs. We then applied this adipose-EP protocol to deliver a DNA vaccine coding for an influenza antigen into guinea pigs. The resulting host immune responses elicited were of a similar magnitude to those achieved by skin delivery with EP. The onset of the humoral immune response was more rapid when the DNA dose was spread over multiple injection sites, and increasing the voltage of the EP device increased the magnitude of the immune response. This study supports further development of EP protocols delivering gene-based therapies to subcutaneous fat.

An electroporation-free method based on Red recombineering for markerless deletion and genomic replacement in the Escherichia coli DH1 genome.

The λ-Red recombination system is a popular method for gene editing. However, its applications are limited due to restricted electroporation of DNA fragments. Here, we present an electroporation-free λ-Red recombination method in which target DNA fragments are excised by I-CreI endonuclease in vivo from the landing pad plasmid. Subsequently, the I-SceI endonuclease-cutting chromosome and DNA double-strand break repair were required. Markerless deletion and genomic replacement were successfully accomplished by this novel approach. Eight nonessential regions of 2.4–104.4 kb in the Escherichia coli DH1 genome were deleted separately with selection efficiencies of 5.3–100%. Additionally, the recombination efficiencies were 2.5–45%, representing an order of magnitude improvement over the electroporation method. For example, for genomic replacement, lycopene expression flux (3.5 kb) was efficiently and precisely integrated into the chromosome, accompanied by replacement of nonessential regions separately into four differently oriented loci. The lycopene production level varied approximately by 5- and 10-fold, corresponding to the integrated position and expression direction, respectively, in the E. coli chromosome.

A rapid seamless method for gene knockout in Pseudomonas aeruginosa

BackgroundPseudomonas aeruginosa is a model organism for the study of quorum sensing, biofilm formation, and also leading cause of nosocomial infections in immune compromised patients. As such P. aeruginosa is one of the most well studied organisms in terms of its genetics. However, the construction of gene deletions and replacements in Pseudomonas aeruginosa is relatively time-consuming, requiring multiple steps including suicide vector construction, conjugation, inactivation with insertion of antibiotic resistance cassettes and allelic exchange. Even employing Gateway recombineering techniques with direct transformation requires a minimum two weeks.MethodsWe have developed a rapid streamlined method to create clean deletion mutants in P. aeruginosa through direct transformation, eliminating the need for the creation of Gateway-compatible suicide vectors. In this method, upstream and downstream sequences of the gene/locus to be deleted are amplified by polymerase chain reaction (PCR) and seamlessly fused with the linearized pEX18Tc sacB suicide plasmid by Gibson assembly. The resulting deletion plasmid is transformed into P. aeruginosa by an electroporation method optimized in this study. The plasmid is then integrated into the chromosome by homologous recombination, and deletion mutants are identified via sacB mediated sucrose counter-selection.ResultsThe current method was employed to generate clean gene deletions of the heme assimilation system anti-σ factor, hasS and the virulence regulator involving ECF system anti-σ and σ factors vreA and vreI, respectively. The process from plasmid construction to confirmation by DNA sequencing of the gene deletion was completed in one week. Furthermore, the utility of the method is highlighted in the construction of the vreA and vreI deletions, where the start codon of vreA and the stop codon of vreI overlap. Utilizing Gibson assembly deletion mutants were constructed with single base pair precision to generate the respective vreA and vreI deletions, while maintaining the start and stop codon of the respective genes. Overall, this method allows for rapid construction of gene deletions in P. aeruginosa with base pair precision.ConclusionThis method from the construction of the suicide vector to sequence confirmation of the unmarked gene deletion can be performed in one week, without the requirement for expensive proprietary reagents or instruments. The precision of Gibson assembly and the fact the accuracy in generating the desirable construct is 95%, makes this a viable and attractive alternative to previous methods.

Hit-and-run programming of therapeutic cytoreagents using mRNA nanocarriers

Therapies based on immune cells have been applied for diseases ranging from cancer to diabetes. However, the viral and electroporation methods used to create cytoreagents are complex and expensive. Consequently, we develop targeted mRNA nanocarriers that are simply mixed with cells to reprogram them via transient expression. Here, we describe three examples to establish that the approach is simple and generalizable. First, we demonstrate that nanocarriers delivering mRNA encoding a genome-editing agent can efficiently knock-out selected genes in anti-cancer T-cells. Second, we imprint a long-lived phenotype exhibiting improved antitumor activities into T-cells by transfecting them with mRNAs that encode a key transcription factor of memory formation. Third, we show how mRNA nanocarriers can program hematopoietic stem cells with improved self-renewal properties. The simplicity of the approach contrasts with the complex protocols currently used to program therapeutic cells, so our methods will likely facilitate manufacturing of cytoreagents.

Transformation of Schizosaccharomyces japonicus.

This protocol describes the use of electroporation to transform Schizosaccharomyces japonicus with plasmids or linear DNA. Plasmids are helpful for the complementation testing of mutations and for the expression of specific genes. Linear DNA fragments integrated into chromosomal DNA by homologous recombination are useful for gene deletion or to fuse a gene with a tag sequence (e.g., encoding a fluorescent protein). To introduce DNA into S. japonicus, electroporation methods are recommended because S. japonicus is sensitive to lithium acetate (LiOAc).

Influence of Different Pulse Length towards Motility and Viability of Ornamental Japanese Carp (Cyprinus carpio Var. Koi) Sperm through Electroporation Method

Successful studies about implementation of electroporation method which sperm becomes gene transfer medium for fish transgenesis have reported. Motility and viability are two major factors in successful of electroporation. The objective of the study was to determine optimal pulse length of electroporation towards the motility and viability of Koi fish sperm. The study was conducted at the Central Laboratory of Biological Sciences and Reproduction of Brawijaya University. The data analysis was conducted using completely randomized design. The electroporation method was carried out using voltage of 30 volt with pulse number of 4 times. The pulse length of the electroporation (based on treatment) was 0.5, 1 and 1.5 ms. The result showed the highest percentage of motility was 3.67% in treatment A (pulse length 0.5 ms) with coefficient correlation R 2 = 0.9643 and the best percentage of viability was 79.67% in treatment A (pulse length 0.5 ms) with coefficient correlation R 2 = 1. Correlation between the pulse length treatment (electroporation) toward motility and viability of Koi fish sperm, in which longer duration of pulse length treatment (electroporation) would decrease the motility and viability percentage of the fish sperm. Keywords: Electroporation, Motility, Pulse length, Sperm, Viability.

The Effect of Electroporation Method towards the Motility and Viability of Java Barb Fish (Puntius javanicus) Sperm

Electric shock treatment of Java Barb Fish ( Puntius javanicus ) sperm using electroporation method on sperm as transfer gen (Sperm Mediated Gen Transfer) has not been implemented in Indonesia. This study was conducted to know the effect of electric shock using gene pulser at different voltage level toward motility and viability of Java barb fish sperm. This research was conducted at Fish Breeding Laboratory, Faculty of Marine and Fisheries and LSIH of Brawijaya University-Malang in May 2017. The trial design used Complete Randomized Design with 3 different treatments and 1 control which each treatment repeated 3 times. The treatments used the Gene pulser with 3 different voltages: A (20 V), B (30 V), C (40 V). The result showed that the electric shock treatment with different voltages level affected motility and viability of Java barb fish sperm. Based on the data analysis used polynomial orthogonal, a linear-form of the relationship among the treatments in the form of equation was found ( Puntius javanicus ) with R 2 =0.9815 and equation y = -40.5x + 130.83 with R 2 = 0.8626. Based on the result of this research, electroporation for Java barb fish’ sperm as gene transfer media should be done with voltage 20 V, pulse number 4 times and pulse length 1 ms. Keywords: electroporation, motility, Puntius javanicus, sperm, viability.

Regulation of axon arborization pattern in the developing chick ciliary ganglion: Possible involvement of caspase 3.

During a certain critical period in the development of the central and peripheral nervous systems, axonal branches and synapses are massively reorganized to form mature connections. In this process, neurons search their appropriate targets, expanding and/or retracting their axons. Recent work suggested that the caspase superfamily regulates the axon morphology. Here, we tested the hypothesis that caspase 3, which is one of the major executioners in apoptotic cell death, is involved in regulating the axon arborization. The embryonic chicken ciliary ganglion was used as a model system of synapse reorganization. A dominant negative mutant of caspase-3 precursor (C3DN) was made and overexpressed in presynaptic neurons in the midbrain to interfere with the intrinsic caspase-3 activity using an in ovo electroporation method. The axon arborization pattern was 3-dimensionally and quantitatively analyzed in the ciliary ganglion. The overexpression of C3DN significantly reduced the number of branching points, the branch order and the complexity index, whereas it significantly elongated the terminal branches at E6. It also increased the internodal distance significantly at E8. But, these effects were negligible at E10 or later. During E6-8, there appeared to be a dynamic balance in the axon arborization pattern between the "targeting" mode, which is accompanied by elongation of terminal branches and the pruning of collateral branches, and the "pathfinding" mode, which is accompanied by the retraction of terminal branches and the sprouting of new collateral branches. The local and transient activation of caspase 3 could direct the balance towards the pathfinding mode.

Attempt to Establish Direct Gene Transformation System to Seeds of Sweet Potato (Ipomoea batatas) Using Electroporation Method

Attempt to Establish Direct Gene Transformation System to Seeds of Sweet Potato (Ipomoea batatas) Using Electroporation Method

Doubled Haploid Transgenic Wheat Lines by Microspore Transformation.

Microspores are preferred explant choice for genetic transformation, as their use shortens the duration of obtaining homozygous transformants. All established gene-delivery methods of particle bombardment, electroporation, and cocultivation with Agrobacterium tumefaciens were optimized on androgenic microspores or derived tissues. In the biolistic gene delivery method 35-40days old haploid microspore embryoids were used for genetic transformation, whereas freshly isolated androgenic microspores were used for genetic transformation in the electroporation and Agrobacterium cocultivation-based methods. The genetic transformation methods of biolistic gene-delivery and electroporation gave rise to the chimeric plants, whereas the method involving cocultivation with Agrobacterium yielded homozygous transformants. These methods were tested on a large number of cultivars belonging to different market classes of wheat, and found to be fairly independent of the explant genotype. Other benefits of using microspores or derived tissues for transformation are: (1) a few explant donors are required to obtain desired transformants and (2) the time required for obtaining homozygous transformants is about 8months in case of spring wheat genotypes and about a year in case of winter wheat genotypes.

Cloning of Lipase Gene From Thermomyces langinosus into Pichia pastoris with its Original Signal Peptide

Lipase is one of the most important industrial enzymes, which is widely used in the preparation of food additives, cosmetics, and pharmaceutical industries. In the previous study, we have cloned synthetic Thermomyces lanuginosus lipase gene into Bacillus subtilis and Escherichia coli and resulting low expression for enzyme activity. The aim of this research was to construct the Thermomyces lanuginosus lipase (TLL) gene into Pichia pastoris vector expression with TLL original signal peptide. TLL gene was amplified by PCR and contained original signal peptide and then inserted into pPICZα A between XhoI and XbaI site, and transformed into competent cell E.coli DH5α. From the transformant, two of positive recombinants were analyzed by sequencing analysis. As the result, both of two recombinant have a positive target gene which has lipase gene. The correct plasmid was linearized and then was transformed in Pichia pastoris X-33 by electroporation method. Thermomyces lanuginosus synthetic gene lipase has been successfully integrated into chromosome of P. pastoris X-33, which revealed by clear zones arund the colony on Yeast extract Peptone Dextrose Tributyrin (YPD.TB) plate with zeocin. The Thermomyces lanuginosus lipase had an open reading frame of 916bp encoding TLL of 314 amino acids with theoretical molecular mass of 35 kDa. The recombinant enzyme, Thermomyces lanuginosus lipase had optimal temperature at 80˚C and optimal pH at pH 8.

Possibilities of the method of irreversible electroporation in treatment of the local and widespread pancreatic cancer

Methods of a local destruction of tumors didn’t find till today wide circulation in treatment of patients with formations of the pancreas (P). It is bound to features of blood supply, anatomical and histological structure of PZh, and also a large number of complications and a recurrence.The technique of the irreversible elektroporation (IE) represents a new unique method of not thermal ablyation at which impact on tissues is carried out by short electric impulses of high voltage (to 3 kV) that involves irreversible rising of permeability of cellular membranes and death of cells. The optimum mechanism of rising of permeability of a cellular membrane electric impulses concerning their frequency or repetitions is yet not up to the end clear; it is recognized that outcomes depend on amplitude, duration and number of impulses. Influence has to be synchronized with a cordial rhythm in order to avoid development of an arrhythmia. Existence at the patient of an arrhythmia and the artificial driver of a rhythm is contraindication to carrying out an elektroporation.We have experience of use of a technique for 18 patients with a locally-spread pancreatic cancer. It was succeeded to reflect the main advantages of a new technique in our experience, such as comparative simplicity of workmanship, safety for vascular and pro-current structures, the minimum impact on function of a pancreas.Efficiency of a destruction is proved to ultrasonic, computer and morphological researches in the postoperative period. The maximum term of observation made 19 months.Though exact indications to use of NE still accurately aren’t defined, the international experience and the first own data allow to state efficiency of new technology in palliative surgical treatment of perivascular tumors of a pancreas, and also its safety.

Transfecting CHO-K1 Cells: Comparison of CaPO4, Electroporation and Lipoplex Method with In-house Prepared Polyplex

The extensive demand of large scale therapeutic protein production in Chinese Hamster Ovarian cell lines increased the need of developing efficient and cost effective transfection reagent. Polyethylenimine has recently emerged as an attractive alternative to currently employed conventional transfection methods. In the present report, we attempted to develop inexpensive and easy to prepare polyethylenimine-based transfection reagent and compared its transfection efficiency with calcium phosphate, electroporation and lipoplex mediated transfection methods. Our results indicated highest transfection efficiency with in-house prepared polyplexes as marked by maximum percentage of enhanced cyan fluorescence protein positive cells. These findings contribute towards the development of economical and effective solution for high protein expression in Chinese hamster ovarian system.

In Ovo Electroporation Methods in Chick Embryos.

To elucidate a gene function, in vivo analysis is indispensable. We can carry out gain and loss of function experiment of a gene of interest by electroporation in ovo and ex ovo culture system on early-stage and advanced-stage chick embryos, respectively. In this section, we introduce in/ex ovo electroporation methods for the development of the chick central nervous system and visual system investigation.

Genome Editing of Rat.

Many genetically engineered rat strains have been produced for biomedical research. The simple and quick production of knock-out and knock-in rats is currently possible using genome editing techniques incorporating zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), or clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Genome-edited animals have been produced by the introduction of endonucleases into embryos using conventional microinjection and a new electroporation method named Technique for Animal Knockout system by Electroporation (TAKE). This chapter introduces the latest protocols for producing genetically engineered rats using genome editing.

Development of an efficient electroporation method for rhizobacterial Bacillus mycoides strains

In order to develop a method for electroporation of environmental Bacillus mycoides strains, we optimized several conditions that affect the electroporation efficiency of this bacterium. By combining the optimized conditions, the electroporation efficiency of strain EC18 was improved to (1.3±0.6)×105cfu/μg DNA, which is about 103-fold increase in comparison with a previously reported value. The method was further validated on various B. mycoides strains, yielding reasonable transformation efficiencies. Furthermore, we confirmed that restriction/modification is the main barrier for electroporation of this bacterium. To the best of our knowledge, this is the first systematic investigation of various parameters of electroporation of B. mycoides. The electroporation method reported will allow for efficient genetic manipulation of this bacterium.

A Thermographic Comparison of Irreversible Electroporation and Radiofrequency Ablation

Radiofrequency ablation and irreversible electroporation are both interventional procedures used in treating cancer diseases generally and may be used in treatment of liver cancer. The two differ in terms of their action and thermal effects. The present study examined thermal effects of radiofrequency ablation and modified irreversible electroporation ex vivo in porcine liver. Standard radiofrequency ablation was used during the experiments, as was a unique, experimental high-voltage generator of the research team's own construction coupled with a newly designed balloon electrode catheter for irreversible electroporation. The tested balloon catheter was newly designed for application in the biliary tract. The temperature of porcine liver tissue was monitored without contact by infrared camera. The results show significantly different temperature of tissue around electrodes for radiofrequency ablation and irreversible electroporation. The data obtained from radiofrequency ablation with maximum tested output power of 10 watts show a symmetrical area of tissue near active electrodes with temperature increased by more than 30 °C. Irreversible electroporation methods showed temperature increasing approximately by 5 °C. To summarize, the thermal research showed that the difference between standard RFA and innovated IRE with the balloon catheter lies not just in the differing mechanism of action, as is well known, but also in its thermal effects. There was documented that tissue temperature was much higher for RFA than for IRE, particularly for the maximum tested output parameters. The applicability of the newly designed balloon catheters was confirmed.

Role of Class III phosphoinositide 3-kinase in the brain development: possible involvement in specific learning disorders.

Class III phosphoinositide 3-kinase (PIK3C3 or mammalian vacuolar protein sorting 34 homolog, Vps34) regulates vesicular trafficking, autophagy, and nutrient sensing. Recently, we reported that PIK3C3 is expressed in mouse cerebral cortex throughout the developmental process, especially at early embryonic stage. We thus examined the role of PIK3C3 in the development of the mouse cerebral cortex. Acute silencing of PIK3C3 with in utero electroporation method caused positional defects of excitatory neurons during corticogenesis. Time-lapse imaging revealed that the abnormal positioning was at least partially because of the reduced migration velocity. When PIK3C3 was silenced in cortical neurons in one hemisphere, axon extension to the contralateral hemisphere was also delayed. These aberrant phenotypes were rescued by RNAi-resistant PIK3C3. Notably, knockdown of PIK3C3 did not affect the cell cycle of neuronal progenitors and stem cells at the ventricular zone. Taken together, PIK3C3 was thought to play a crucial role in corticogenesis through the regulation of excitatory neuron migration and axon extension. Meanwhile, when we performed comparative genomic hybridization on a patient with specific learning disorders, a 107 Kb-deletion was identified on 18q12.3 (nt. 39554147-39661206) that encompasses exons 5-23 of PIK3C3. Notably, the above aberrant migration and axon growth phenotypes were not rescued by the disease-related truncation mutant (172 amino acids) lacking the C-terminal kinase domain. Thus, functional defects of PIK3C3 might impair corticogenesis and relate to the pathophysiology of specific learning disorders and other neurodevelopmental disorders. Acute knockdown of Class III phosphoinositide 3-kinase (PIK3C3) evokes migration defects of excitatory neurons during corticogenesis. PIK3C3-knockdown also disrupts axon outgrowth, but not progenitor proliferation invivo. Involvement of PIK3C3 in neurodevelopmental disorders might be an interesting future subject since a deletion mutation in PIK3C3 was detected in a patient with specific learning disorders (SLD).

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation.

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

<em>In Vivo</em> Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.