Efficient in situ electroporation of mammalian cells grown on microporous membranes.

Electroporation is a common technique for the introduction of DNA molecules into living cells. The method is currently limited by the necessity of applying the electrical discharge to cells in suspension. Adherent cells must therefore be removed from their substratum, which can induce unwanted physiological effects. We report here a new procedure for in situ electroporation of cells grown on microporous membranes of polyethylene terephthalate (PET) or polyester (PE). We demonstrate that this method of in situ electroporation employs only readily available materials and standard electroporation devices without any modifications, is as efficient as conventional electroporation of cells in suspension, and is applicable to a wide range of cell types. Efficient electroporation can be achieved under conditions of minimal cell killing, and can be performed with quiescent cells as well as with confluent epithelial sheets. The method is a useful extension of electroporation technology, and will allow the application of electroporation to a wider spectrum of biological systems.     

银胶浓度对电穿孔获取细胞内表面增强拉曼光谱的影响

探究了银胶浓度对于电穿孔导入银纳米粒子获取细胞内表面增强拉曼光谱(SERS)的影响.对6组含有不同浓度银胶的鼻咽癌细胞C666进行电穿孔,测量电穿孔后活细胞内表面增强拉曼光谱.以测得的SERS信号、光谱强度积分值和谱线重复性为指标,研究银胶浓度对电穿孔获取细胞内SERS的影响,对电穿孔后活性C666细胞内SERS平均光谱进行初步谱峰归属.在脉冲电场强度875 V/cm,脉冲持续时间1 ms,电脉冲2次的条件下,每500μl电击缓冲液中含有50μl银胶时测得的细胞内SERS光谱信噪比高,且光谱具有较好的重复性.结果说明,正确选择银胶浓度可以提高电穿孔-SERS效果,获取高质量的活细胞内SERS信号.此研究有助于扩展表面增强拉曼光谱的应用,包括实时检测分析活细胞内生化成分及分布,实时监测细胞生化变化过程等.     

Electroporation of heterogeneous lipid membranes

Electroporation is the basis for the transfection of genetic material and for drug delivery to cells, including electrochemotherapy for cancer. By means of molecular dynamics many aspects of membrane electroporation have been unveiled at the molecular detail in simple, homogeneous, lipid bilayers. However, the correspondence of these findings \with the process happening in cell membranes requires, at least, the consideration of laterally structured membranes. Here, I present a systematic molecular dynamics study of bilayers composed of different liquid-ordered and liquid-disordered lipid phases subjected to a transversal electric field. The simulations reveal two significant results. First, the electric field mainly affects the properties of the disordered phases, so that electroporation takes place in these membrane regions. Second, the smaller the disordered domains are, the faster they become electroporated. These findings may have a relevant significance in the experimental application of cell electroporation in vivo since it implies that electro-induced and pore-mediated transport processes occur in particularly small disordered domains of the plasma membrane, thus locally affecting only specific regions of the cell.     

Electroporation: A general phenomenon for manipulating cells and tissues

Electroporation is a fascinating cell membrane phenomenon with several existing biological applications and others likely. Although DNA introduction is the most common use, electroporation of isolated cells has also been used for (1) introduction of enzymes, antibodies, and other biochemical reagents for intracellular assays; (2) selective biochemical loading of one size cell in the presence of many smaller cells; (3) introduction of virus and other particles; (4) cell killing under nontoxic conditions; and (5) insertion of membrane macromolecules into the cell membrane. More recently, tissue electroporation has begun to be explored, with potential applications including (1) enhanced cancer tumor chemotherapy, (2) gene therapy, (3) transdermal drug delivery, and (4) noninvasive sampling for biochemical measurement. As presently understood, electroporation is an essentially universal membrane phenomenon that occurs in cell and artificial planar bilayer membranes. For short pulses (μs to ms), electroporation occurs if the transmembrane voltage, U(t), reaches 0.5–1.5 V. In the case of isolated cells, the pulse magnitude is 10³–10⁴ V/cm. These pulses cause reversible electrical breakdown (REB), accompanied by a tremendous increase molecular transport across the membrane. REB results in a rapid membrane discharge, with the elevated U(t) returning to low values within a few microseconds of the pulse. However, membrane recovery can be orders of magnitude slower. An associated cell stress commonly occurs, probably because of chemical influxes and effluxes leading to chemical imbalances, which also contribute to eventual survival or death. Basic phenomena, present understanding of mechanism, and the existing and potential applications are briefly reviewed.     

电穿孔技术在转基因及动物克隆中的应用

电穿孔技术利用电场造成细胞膜的改变而将DNA导入细胞内,它还可用于细胞融合及动物克隆等。基因电转移的效率通常比化学法提高1—2个数量级,主要与脉冲波形、长度、缓冲液等有关。方波直流电脉冲应用广泛,在有关细胞核移植的多项研究报告中均指出它有重要作用。     

Introduction of deoxyribonucleoside triphosphates into intact cells by electroporation

A simple method, employing high-voltage electric discharge (electroporation), was developed to introduce phosphorylated nucleosides into the cytoplasm of viable cells. HL-60 leukemia cells permeabilized by this technique remained viable and incorporated deoxyribonucleoside triphosphates into nuclear DNA. Furthermore, DNA synthesis was depressed for at least 24 h in HL-60 cells made permeable to 1-beta-D-arabinosylcytosine 5'-triphosphate by this methodology. Electroporation was found to be applicable to the permeabilization of a wide variety of cell lines in culture to nucleotides, suggesting that this methodology may be useful for the introduction into intact cells of a wide variety of molecules that are not normally transported effectively.     

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons¹, which has been difficult with the conventional method of injection and electroporation at E1.5².     

Genetic transformation of Streptococcus thermophilus by electroporation

A rapid and convenient electroporation procedure was developed for the genetic transformation of intact cells of Streptococcus thermophilus with various species of plasmid DNA. Transformation frequency was influenced by the capacitance and voltage selected for electric pulsing, the pH and composition of the electroporation medium and the molecular mass of the transforming DNA. Electroporation is a simple and effective technique to introduce plasmid DNA into S. thermophilus and useful in the development of recombinant DNA technology for this important industrial microorganism.     

Gene transfer into mouse lyoma cells by electroporation in high electric fields.

Electric impulses (8 kV/cm, 5 microseconds) were found to increase greatly the uptake of DNA into cells. When linear or circular plasmid DNA containing the herpes simplex thymidine kinase (TK) gene is added to a suspension of mouse L cells deficient in the TK gene and the cells are then exposed to electric fields, stable transformants are formed that survive in the HAT selection medium. At 20 degrees C after the application of three successive electric impulses followed by 10 min to allow DNA entry there result 95 (+/- 3) transformants per 10(6) cells and per 1.2 micrograms DNA. Compared with biochemical techniques, the electric field method of gene transfer is very simple, easily applicable, and very efficient. Because the mechanism of DNA transport through cell membranes is not known, a simple physical model for the enhanced DNA penetration into cells in high electric fields is proposed. According to this ' electroporation model' the interaction of the external electric field with the lipid dipoles of a pore configuration induces and stabilizes the permeation sites and thus enhances cross membrane transport.     

Electroporating Fields Target Oxidatively Damaged Areas in the Cell Membrane

Reversible electropermeabilization (electroporation) is widely used to facilitate the introduction of genetic material and pharmaceutical agents into living cells. Although considerable knowledge has been gained from the study of real and simulated model membranes in electric fields, efforts to optimize electroporation protocols are limited by a lack of detailed understanding of the molecular basis for the electropermeabilization of the complex biomolecular assembly that forms the plasma membrane. We show here, with results from both molecular dynamics simulations and experiments with living cells, that the oxidation of membrane components enhances the susceptibility of the membrane to electropermeabilization. Manipulation of the level of oxidative stress in cell suspensions and in tissues may lead to more efficient permeabilization procedures in the laboratory and in clinical applications such as electrochemotherapy and electrotransfection-mediated gene therapy.     

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.     

Transfection of cells using flow-through electroporation based on constant voltage

Electroporation is a high-efficiency and low-toxicity physical gene transfer method. Classical electroporation protocols are limited by the small volume of cell samples processed (less than 10(7) cells per reaction) and low DNA uptake due to partial permeabilization of the cell membrane. Here we describe a flow-through electroporation protocol for continuous transfection of cells, using disposable devices, a syringe pump and a low-cost power supply that provides a constant voltage. We show transfection of cell samples with rates ranging from 40 μl min(-1) to 20 ml min(-1) with high efficiency. By inducing complex migrations of cells during the flow, we also show permeabilization of the entire cell membrane and markedly increased DNA uptake. The fabrication of the devices takes 1 d and the flow-through electroporation typically takes 1-2 h.     

Numerical modeling of in vivo plate electroporation thermal dose assessment

Electroporation is an approach used to enhance the transport of large molecules to the cell cytosol in which a targeted tissue region is exposed to a series of electric pulses. The cell membrane, which normally acts as a barrier to large molecule transport into the cell interior, is temporarily destabilized due to the development of pores in the cell membrane. Consequently, agents that are ordinarily unable enter the cell are able to pass through the cell membrane. Of possible concern when exposing biological tissue to an electric field is thermal tissue damage associated with joule heating. This paper explores the thermal effects of various geometric, biological, and electroporation pulse parameters including the blood vessel presence and size, plate electrode configuration, and pulse duration and frequency. A three-dimensional transient finite volume model of in vivo parallel plate electroporation of liver tissue is used to develop a better understanding of the underlying relationships between the physical parameters involved with tissue electroporation and resulting thermal damage potential.     

Changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy.

Cells can be transiently permeabilized by exposing them briefly to an intense electric field (a process called "electroporation"), but it is not clear what structural changes the electric field induces in the cell membrane. To determine whether membrane pores are actually created in the electropermeabilized cells, rapid-freezing electron microscopy was used to examine human red blood cells which were exposed to a radio-frequency electric field. Volcano-shaped membrane openings appeared in the freeze-fracture faces of electropermeabilized cell membranes at intervals as short as 3 ms after the electrical pulse. We suggest that these openings represent the membrane pathways which allow entry of macromolecules (such as DNA) during electroporation. The pore structures rapidly expand to 20-120 nm in diameter during the first 20 ms of electroporation, and after several seconds begin to shrink and reseal. The distribution of pore sizes and pore dynamics suggests that interactions between the membrane and the submembrane cytoskeleton may have an important role in the formation and resealing of pores.     

An improved method of electroporation for introducing biologically active foreign genes into cultured mammalian cells

We have developed a modified, reproducible, and efficient method for introducing cloned genes into mammalian cells by using an electric field followed by treatment with sodium butyrate. Transfection frequencies with plasmid pSV2-neo, consisting of an antibiotic (G418) resistance gene and simian virus 40 (SV40) early promoter, by electroporation were higher than those by calcium phosphate DNA precipitation. Treatment with sodium butyrate following electroporation significantly increased the frequency of transfection in various types of cell lines and primary cultured cells including human skin fibroblasts. Treatment with sodium butyrate also increased the transient expression of the gene for chloramphenicol acetyltransferase (acetyl-CoA; chloramphenicol O3-acetyltransferase, CAT, EC 2.3.1.28) when the gene was introduced into BALB/c 3T3 cells by electroporation. Electroporation combined with sodium butyrate treatment is an improved method for stable and transient biochemical transformation of foreign genes in cultured mammalian cells.     

Use of electroporation for high-molecular-weight DNA-mediated gene transfer

Electroporation was used to introduce high-molecular-weight DNA into murine hematopoietic cells and NIH3T3 cells. CCRF-CEM cells were stably transfected with SV2NEO plasmid and the genomic DNA from G-418-resistant clones (greater than 65 kb) was introduced into mouse bone marrow and NIH3T3 cells by electroporation. NEO sequences and expression were detected in the hematopoietic tissues of lethally irradiated mice, with 24% of individual spleen colonies expressing NEO. The frequency of genomic DNA transfer into NIH3T3 cells was 0.25 X 10(-3). Electroporation thus offers a powerful mode of gene transfer not only of cloned genes but also of high-molecular-weight DNA into cells.     

Introduction of large molecules into viable fibroblasts by electroporation: optimization of loading and identification of labeled cellular compartments.

Access to the cell cytoplasm in viable cells may permit direct labeling or manipulation of intracellular molecules and metabolic processes. One method to gain access to the cell cytoplasm is by electroporation, a technique that transiently creates pores in cell membranes by means of applied electrical fields. We used electroporation to introduce large-molecular-mass dextrans and proteins as probes of the cytoplasmic compartment in human gingival fibroblasts. Electrical field strength and pulse decay time were optimized to obtain cellular viability greater than 80%. Analysis by confocal microscopy and by fluorescence spectrophotometry demonstrated that a large proportion of high-molecular-mass probe was membrane-bound after electroporation. Trypsinization did not affect membrane-bound FITC-dextran but eliminated protein probe incorporated into the membrane, thereby permitting measurement of only intracellular, cytoplasmic label. Proteins of up to 66 kDa were incorporated at intracellular concentrations of 10(-15) M. After electroporation under optimal conditions, incorporated anti-vimentin antibodies were capable of binding to vimentin. Cells electroporated in the presence of RNase A exhibited significant reductions of cellular RNA. Electroporation appears to be a useful approach to probe or perturb specific cellular processes by introduction of functional molecular species into the cytoplasm of viable 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.