Introduction of antibody into viable cells using electroporation

Conditions for labelling an intracellular antigen, p21ras, using electroporation to introduce a fluorescent antibody, are described. Following labelling, cells were evaluated for p21ras associated fluorescence by flow cytometry. Electroporation, sorting, and cell handling parameters were varied to determine optimal conditions for cell viability. Cells were best held in serum containing growth medium both before and after electroporation, while antibody introduction during the electroporation phase was most efficient when carried out in a balanced saline solution. For maximum efficiency of antibody internalization, the antibody needed to be present during electroporation, and medium needed to be replaced several times in the first few hours after electroporation to ensure good cell survival.     

Optimization of Electrotransfection Conditions of Mammalian Cells with Different Biological Features

We introduced eukaryotic expression plasmid pEGFP-N1 encoding green fluorescent protein (GFP) genes into cells with different biological features through electroporation. The effects of conditions, including voltage, capacitor flow, pulse cycle, DNA dosage and buffer, on transfection efficiency were investigated based on fluorescent microscopy and posttransfection survival rate of cells by staining with trypan blue. Better electrotransfection outcomes were achieved in the following epithelial cells: Vero cells at 300?V/850???F, PK15 cells at 300?V/500???F, MDCK cells at 200?V/600???F, F81 cells at 200?V/500???F, cancer cells MB49 at 300?V/400???F, Hela cells at 200?V/450???F, HF-29 cells at 300?V/800???F and B16F1 cells at 200?V/650???F. Among fibroblast cells, better electrotransfection was achieved in BHK21 cells at 300?V/600???F and ST cells at 200?V/750???F. RPMI-1640 medium without antibiotics and serum demonstrated higher electrotransfection efficiency and cell survival rate than other cell culture media as electroporation buffer. Our findings further prove that electroporation transfection is an effective method for genetic transfection. Cells with different biological features require varying transfection conditions to obtain higher transfection efficiency of target genes.     

一种无血清冻存液在猪胎儿成纤维细胞上的应用研究

在猪胎儿成纤维细胞(porcine fetal fibroblasts, PFF)冻存过程中,血清品质常常制约着细胞的冻存效果。为了解决这个问题,本研究旨在开发一种无血清冻存液应用于猪胎儿成纤维细胞冻存。用3种不同冻存液冻存猪胎儿成纤维细胞,每种冻存10管。冻存30 d后复苏细胞,测定冻存细胞存活率,细胞增殖活力以及电转后细胞活性。结果显示:自制无血清细胞冻存液,冻存猪胎儿成纤维细胞后存活率达95.33%;细胞增殖活力以及电转后细胞活性均显著高于标准胎牛血清冻存液(p<0.05),与特级胎牛血清冻存液效果相当(p>0.05)。因此,自制冻存液冻存猪胎儿成纤维细胞效果稳定,能够替代含血清冻存液,有良好的推广应用前景。     

Use of a transient expression assay for the optimization of direct gene transfer into tobacco mesophyll protoplasts by electroporation

Plasmid DNA was transfected into tobacco mesophyll protoplasts by electroporation. Transfection efficiency was estimated, using a transient expression assay based on the measurement of chloramphenicol transacetylase activity or by scoring colonies expressing resistance to paromomycin, an aminoglycoside related to kanamycin. Under conditions of cell survival superior to 50% after electroporation, transient expression signals and transformation efficiencies were found to be proportional. Factors affecting the efficiency of transformation were studied. A clear-cut optimum voltage (250-300 V/cm) was detected. Among various salts tested, potassium chloride was the best electrolyte. No improvement of electroporation efficiency was obtained by a heat-shock (45 degrees C/5 min) treatment prior to electroporation or by the presence of polyethylene glycol in the electroporation medium. The physiological state of plants used as the protoplast source significantly affected the transfection ability of the resulting protoplasts. These results are discussed and compared to previously published procedures.     

Efficient transfection of primarily cultured porcine embryonic fibroblasts using the Amaxa Nucleofection system

Porcine embryonic fibroblasts (PEF) are important as donor cells for nuclear transfer for generation of genetically modified pigs. In this study, we determined an optimal protocol for transfection of PEF with the Amaxa Nucleofection system, which directly transfers DNA into the nucleus of cells, and compared its efficiency with conventional lipofection and electroporation. Cell survival and transfection efficiency were assessed using dye-exclusion assay and a green fluorescent protein (GFP) reporter construct, respectively. Our optimized nucleofection parameters yielded survival rates above 60%. Under these conditions, FACS analysis demonstrated that 79% of surviving cells exhibited transgene expression 48 h after nucleofection when program U23 was used. This efficiency was higher than that of transfection of PEFs with electroporation (ca. 3-53%) or lipofection (ca. 3-8%). Transfected cells could be expanded as stably transgene-expressing clones over a month. When porcine nuclear transfer (NT) was performed using stable transformant expressing GFP as a donor cell, 5-6% of reconstituted embryos developed to blastocysts, from which 30-50% of embryos exhibited NT-embryo-derived green fluorescence. Under the conditions evaluated, nucleofection exhibited higher efficiency than conventional electroporation and lipofection, and may be a useful alternative for generation of genetically engineered pigs through nuclear transfer.     

Me2SO- and serum-free cryopreservation of human umbilical cord mesenchymal stem cells using electroporation-assisted delivery of sugars

Cryopreservation is the universal technology used to enable long-term storage and continuous availability of cell stocks and tissues for regenerative medicine demands. The main components of standard freezing media are dimethyl sulfoxide (hereinafter Me₂SO) and fetal bovine serum (FBS). However, for manufacturing of cells and tissue-engineered products in accordance with the principles of Good Manufacturing Practice (GMP), current considerations in regenerative medicine suggest development of Me₂SO- and serum-free biopreservation strategies due to safety concerns over Me₂SO-induced side effects and immunogenicity of animal serum.In this work, the effect of electroporation-assisted pre-freeze delivery of sucrose, trehalose and raffinose into human umbilical cord mesenchymal stem cells (hUCMSCs) on their post-thaw survival was investigated. The optimal strength of electric field at 8 pulses with 100 μs duration and 1 Hz pulse repetition frequency was determined to be 1.5 kV/cm from permeabilization (propidium iodide uptake) vs. cell recovery data (resazurin reduction assay).Using sugars as sole cryoprotectants with electroporation, concentration-dependent increase in cell survival was observed. Irrespective of sugar type, the highest cell survival (up to 80%) was achieved at 400 mM extracellular concentration and electroporation. Cell freezing without electroporation yielded significantly lower survival rates. In the optimal scenario, cells were able to attach 24 h after thawing demonstrating characteristic shape and sugar-loaded vacuoles. Application of 10% Me₂SO/90% FBS as a positive control provided cell survival exceeding 90%. Next, high glass transition temperatures determined for optimal concentrations of sugars by differential scanning calorimetry (DSC) suggest the possibility to store samples at −80 °C. In summary, using electroporation to incorporate cryoprotective sugars into cells is an effective strategy towards Me₂SO- and serum-free cryopreservation and may pave the way for further progress in establishing clinically safe biopreservation strategies for efficient long-term biobanking of cells.     

Assessment of electroporation by flow cytometry

BACKGROUND: Electroporation accomplishes transient permeabilization of cells and thus aids in the uptake of drugs. The method has been employed clinically in the treatment of dermatological tumors with bleomycin. The conditions of electroporation are still largely empirical and information is lacking as to the interrelationships among voltage pulse height, pulse number and toxicity, cell permeation, drug uptake, and effects on drug toxicity. We used propidium iodide (PI) and flow cytometry to define cell permeation into cytoplasmic and nuclear compartments to determine the improvements of drug toxicity that can be accomplished by electroporation. METHODS: Human squamous carcinoma cells of defined TP53 status and normal human epithelial cells were subjected to electroporation using a square wave pulse generator in the range of 0-5,000 V/cm. Flow cytometry served to establish entry of the drug reporter, PI, into the cytoplasm and nucleus. A dye staining method served to establish cell survival and to determine the toxicity of bleomycin alone, electroporation alone, and electroporation with bleomycin. RESULTS: The electric field intensity (EFI) required to produce 50% permeabilization (EP(50)) is cell type dependent. The EP(50) varied from 1,465 to 2,027 V/cm. An EFI below 900 V/cm is growth stimulatory whereas an EFI in excess of 1,000 V/cm is growth inhibitory. An EFI of 1,000 V/cm is sufficient to increase bleomycin toxicity by a factor of 2-3. A differential electroporation efficiency is observed between normal and tumor cells. CONCLUSIONS: Tumor cells can be targeted preferentially at electroporation voltages where normal cells are less permeable.     

Electroporation optimization to deliver plasmid DNA into dental follicle cells

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

In Vitro Targeted Gene Electrotransfer to Endothelial Cells with Plasmid DNA Containing Human Endothelin-1 Promoter

Development of recombinant DNA technologies has allowed us to create new delivery systems that target specific cell types and that can be used in gene therapy. One of these targets is vascular endothelium because of its important role in tumor angiogenesis. For tumor endothelium-specific targeting, we prepared plasmid DNA encoding green fluorescent protein under the control of human endothelin-1 promoter (pENDO-EGFP), which is specific for endothelial cells. First we determined gene electrotransfer parameters for improved transfection of endothelial cells evaluating different osmolarity of electroporation buffer, voltages of applied electric pulses, and addition of fetal bovine serum immediately after electroporation to the cells for improved transfection and survival. Transfection efficacy of pENDO-EGFP in different endothelial and nonendothelial cell lines was determined next. Gene electrotransfer efficacy was evaluated using three different methods: fluorescence microscopy, fluorescence microplate reader, and flow cytometry. Our results showed that transfection efficacy was higher when cells were prepared in hypoosmolar compared to isoosmolar electroporation buffer. Furthermore, immediate addition of fetal bovine serum to the cells after pulsing also improved gene electrotransfer into target cells. We proved expression of EGFP under the control of human endothelin-1 promoter in endothelial cells, which was also significantly higher compared to nonendothelial cells. Taken together, we successfully constructed pENDO-EGFP, which was specifically expressed in endothelial cells using improved gene electrotransfer parameters.     

A nonuniform electrical field electroporation chamber design

We show an inexpensive design for an electroporation chamber which subjects electroporated cells to a nonuniform electrical field. Our design, which we call an electroporation cylinder, improved transfection efficiency over that of a uniform field design (electroporation cuvettes) by about sixfold when tested in five mouse cell lines with a transient gene expression assay. Electroporation cylinders subjected cells to electrical field strengths at least as powerful as those of electroporation cuvettes, as judged by comparing the percentages of cells killed by electroporation. Cylinder and cuvette designs were similar in their effect on the variability of transfection efficiency. Electroporation cylinders may be particularly useful when the optimal electrical field strength for a cell line is not known or is unattainable with a given power supply.     

Intradermal delivery of interleukin-12 plasmid DNA by in vivo electroporation

Gene therapy depends on safe and efficient gene delivery. The skin is an attractive target for gene delivery because of its accessibility. Recently, in vivo electroporation has been shown to enhance expression after injection of plasmid DNA. In this study, we examined the use of electroporation to deliver plasmid DNA to cells of the skin in order to demonstrate that localized delivery can result in increased serum concentrations of a specific protein. Intradermal injection of a plasmid encoding luciferase resulted in low levels of expression. However, when injection was combined with electroporation, expression was significantly increased. When performing this procedure with a plasmid encoding interleukin-12, the induced serum concentrations of gamma-interferon were as much as 10 fold higher when electroporation was used. The results presented here demonstrate that electroporation can be used to augment the efficiency of direct injection of plasmid DNA to skin.     

Electroporation of lymphoid cells: factors affecting the efficiency of transfection

We have increased the efficiency of electroporation of lymphoid cells over fifty fold by optimising several biological and electrical parameters. Under optimised conditions, the electroporation efficiency was comparable to that reported for other cell types. Actively dividing cells were crucial for high transient transfection signal. The two most important electrical parameters were high capacitance (960 microF) and moderate decay constants in the range of 10-15 ms. The optimal field strength depended on the cell line, but was in the range 0.6-1 kV/cm. Administering the pulse in medium lacking serum gave higher efficiency than when isotonic salt solution was used and the transfection signal was depressed if cells and DNA were allowed to incubate for several minutes either before or after the pulse. Electroporation was carried out at room temperature and there was no advantage in using low temperatures (0-4 degrees C). When electroporated cells were grown in conditioned medium, the signal was enhanced about two fold depending on the source of the conditioned medium.     

Long duration electroporation for achieving high level expression of glucocorticoid receptors in mammalian cell lines

A method is presented that utilizes long duration electroporation (LDE) to more efficiently introduce DNA into mammalian cell lines than standard electroporation techniques. With SV40-based vectors, more than 550,000 glucocorticoid receptors (GRs) per cell could be obtained in COS-7 cells with good cell survival. In experiments with a CMV-driven vector expressing an enhanced Green Fluorescent Protein (EGFP), 54% of the cells were transfected, and 77% of EGFP positive cells expressed EGFP at moderate to high levels. In cell lines not containing the large T antigen, a CMV-driven vector for the GR was superior to the SV40-based vector. In EDR3, DG44, and CV-1 cell lines approximately 220,000, 190,000 and 150,000 GRs/cell were obtained, respectively. Transfection efficiency of the EGFP vector ranged from 44 to 55% for the three cell lines. Cortisol treatment of COS-7 and DG44 cultures cotransfected with vectors expressing the GR and a GRE driven luciferase gene produced 4 to 12 times more enzyme activity per plate with LDE than conventional electroporation protocols. LDE allows transient overexpression of proteins in COS-7 cells at the high levels generally achieved by mammalian overexpression systems only in stable cell lines.     

Optimization of electroporation for transfection of mammalian cell lines

Electroporation can be a highly efficient method for introducing DNA molecules into cultured cells for transient expression of genes or for permanent genetic modification. However, effective transformation by electroporation requires careful optimization of electric field strength and pulse characteristics. We have used the transient expression of the firefly luciferase gene as a rapid and sensitive indicator of gene expression to describe the effects on transfection efficiency of altering electroporation field strength and shape. Using the luciferase assay, we investigated the correlation of cell viability with optimal transfection efficiency and determined the optimal parameters for a number of phenotypically distinct mammalian cell lines derived from the nervous and immune systems. The efficiency of electroporation under optimal conditions was compared with that obtained using DEAE-dextran or calcium phosphate-mediated transformation. Transfection by electroporation using square wave pulses, as opposed to exponentially decaying pulses, was found to be significantly increased by repetitive pulses. These methods improve the ability to obtain high efficiency gene transfer into many mammalian cell types.     

mRNA Transfection of Mouse and Human Neural Stem Cell Cultures

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.     

Electroporation: parameters affecting transfer of DNA into mammalian cells

Electroporation, the reversible breakdown of cell membranes caused by a high-voltage discharge, is a rapid, simple, and efficient method for introducing DNA into mammalian cells. An instrument for electroporation which permits the high-voltage discharge waveform to be varied with respect to rise time, peak voltage, and fall time is described. The uptake and expression of SV40 DNA following electroporation of two cell types, a human carcinoma-derived cell line, HEp-2, and a human lymphoblastoid cell line, 721, depended on the peak voltage and the fall time of the voltage discharge. The electronic parameters which produced optimum DNA transfer, however, differed for the two cell types. DNA as large as 150 kb was introduced into cells by electroporation. Cells can be electroporated in either phosphate-buffered saline or culture medium containing fetal bovine serum, and the efficiency of DNA transfer does not vary with cell densities from 10(6) to 2 X 10(7)/0.5 ml. Exposing the cells to multiple voltage discharges did not improve DNA transfer. DNA has been introduced by electroporation into all cell types tested, including human carcinoma-derived cell lines, human lymphoblastoid cell lines, human fibroblast strains, and primary human lymphocytes. To obtain maximal DNA transfer by this method, however, one must optimize the peak voltage and fall time of the discharge waveform for each cell type.     

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

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

Gene Transfer to Lentil Protoplasts by Lipofection and Electroporation

Abstract

Cationic liposomes made of dipalmitoylphosphatidylcholine and stearylamine (9:1) were prepared by reverse-phase evaporation and were able to interact spontaneously with plasmid DNA. The loaded vesicles delivered a β-glucuronidase (GUS)-carrying plasmid to lentil Lens culinaris) protoplasts, leading to transient expression of the GUS reporter gene. The transfection efficiency achieved by using stearylamine-containing liposomes (lipofection) was comparable to the one obtained by electroporating the protoplasts at 500 μF and different field strengths. Furthermore, the combination of electroporation and lipofection, though reducing cell survival, increased the activity of the reporter enzyme detected in the cell lysates, yielding transient expression levels higher than those recorded after lipofection or electroporation alone.     

Electrofusion and electroporation of plants

The utilization of electrofusion and electroporation techniques has had a major impact on the genetic manipulation of plants within the last decade. This review of the development of electrofusion and electroporation, as it applies to plants, highlights major developmental aspects of this technology. These include mechanisms for cell fusion, molecular exchange, and parameters that affect the efficiency of fusion and electroporation.