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.     

An electroporation protocol for efficient DNA transfection in PC12 cells

A wide variety of mammalian cell types is used in gene transfection studies. Establishing transfection methods that enable highly efficient DNA uptake has become increasingly important. PC12 is an established rat pheochromocytoma cell line, which responds to exposure to NGF with cessation of growth, expression of cytoplasmic processes, and differentiation into cells resembling sympathetic neurons. Although PC12 cells represent an important model system to study a variety of neuronal functions, they proved relatively difficult to transfect. We have compared the efficiency of three different chemical transfection reagents (Lipofectamine 2000, Lipofectamine LTX and TransIT-LT1) and of two electroporation systems (Neon and Gene Pulser Xcell) in transiently transfecting undifferentiated PC12 cells. By comparing efficiencies from replicate experiments we proved electroporation (in particular Neon) to be the method of choice. By optimizing different parameters (voltage, pulse width and number of pulses) we reached high efficiency of transfection (90 %) and viability (99 %). We also demonstrated that, upon electroporation, cells are not altered by the transfection and maintain their ability to differentiate.     

Optimization of transfection methods for Huh-7 and Vero cells: A comparative study

Availability of an efficient transfection protocol is the first determinant in success of gene transferring studies in mammalian cells which is accomplished experimentally for every single cell type. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for Huh-7 and Vero cells. Different cell confluencies, DNA/reagent ratios and total transfection volumes were optimized for two chemical reagents including jetPEI? and Lipofectamine? 2000. Besides, the effects of electric field strength and pulse length were investigated to improve electroporation efficiency. Transfection of cells by pEGFP-N1 vector and tracking the expression of GFP by FACS and Fluorescence Microscopy analysis were the employed methods to evaluate transfection efficiencies. Optimized electroporation protocols yielded 63.73 ± 2.36 and 73.9 ± 1.6% of transfection in Huh-7 and Vero cells respectively, while maximum achieved level of transfection by jetPEI? was 14.2 ± 0.69 and 28 ± 1.11% Huh-7 and Vero cells, respectively. Post transfectional chilling of the cells did not improve electrotransfection efficiency of Huh-7 cells. Compared to chemical based reagents, electroporation showed superior levels of transfection in both cell lines. The presented protocols should satisfy most of the experimental applications requiring high transfection efficiencies of these two cell lines.     

Apoptosis induced by DNA uptake limits transfection efficiency

Electrotransfection is an effective method for transfecting lymphoid cells. However, the transfection efficiency of certain lymphoid cells is low. L1210 subclones and NFS-70 pro-B cells, which are highly refractory to various transfection methods, were used to identify the limiting factors. Cells were electrotransfected with plasmids coding for green fluorescence protein or luciferase. The luciferase expression of L1210 subclone 3-3 was found to increase 6-12 h after electroporation, but decreased significantly from 12 to 48 h. The lower level of luciferase activity at later time periods correlated with decreases in cell viability, which was shown to be due to apoptosis, as determined by propidium iodide/acrindine orange staining, DNA laddering, and prevention of cell death by addition of caspase inhibitors. Similar results were observed with NFS-70 pro-B cells and select L1210 subclones. In contrast, L1210 parental and L1210 subclone 7-15.6 cells undergo only low levels of apoptosis (< or = 5%). Apoptosis occurred only when DNA (plasmids or salmon sperm DNA) was present during electroporation, but was not dependent on the conformation of the DNA used or the expression of transgenes. Cells pulsed in the presence of dextran sulfate (MW 500,000) did not apoptose. Similar results were observed when L1210 subclone 3-3 was transfected using the cationic lipid 1, 2-dioleoyl-3-trimethylammonium propane, although the transfection efficiency and corresponding rate of apoptosis were significantly lower. Applying the caspase inhibitor fluoromethyl ketone (Boc-ASP-FMK) dramatically improved cell viability and transgene expression of select L1210 subclones and NFS-70 pro-B cells.     

Efficient expression of foreign genes in CHO DHFR cellsby electroporation

DHFR-deficient Chinese hamster ovary (CHO DHFR⁻) cells are the most popular mammalian expression system for inducible amplification of transgene. In order to obtain more stable transfected CHO DHFR⁻ cell clones, transfection efficiency of electroporation under different conditions were systemically investigated using plasmid pSV-β-Gal as reporter gene. Transfection efficiency was proportionally increased with pulse duration and number of pulse applied. In addition, higher transfection efficiency was found in high salt extracellular solution (Berg's and Hank's buffers) than in intracellular solution (cytomix buffer) under the same electroporation condition. The highest transfection efficiency in examined conditions was about 1 in 350 cells (or 0.289%) when cells were electroporated with twice pulses at 400 V, 375 μF. The present study offers an optimized guideline for introducing exogenous DNA into CHO DHFR⁻ cells by electroporation.     

Transfection of bovine fetal fibroblast with polyethylenimine (PEI) nanoparticles: effect of particle size and presence of fetal bovine serum on transgene delivery and cytotoxicity

The development of efficient transfection protocols for livestock cells is crucial for implementation of cell-based transgenic methods to produce genetically modified animals. We synthetized fully deacylated linear 22, 87 and 217 kDa polyethylenimine (PEI) nanoparticles and compared their transfection efficiency and cytotoxicity to commercial branched 25 kDa PEI and linear 58 kDa poly(allylamine) hydrochloride. We studied the effect of PEI size and presence of serum on transfection efficiency on primary cultures of bovine fetal fibroblasts and established cells lines (HEK 293 and Hep G2). We found that transfection efficiency was affected mainly by polymer/pDNA ratio and DNA concentration and in less extent by PEI MW. In bovine fibroblast, preincubation of PEI nanoparticles with fetal bovine serum (FBS) greatly increased percentage of cells expressing the transgene (up to 82%) while significantly decreased the polymer cytotoxic effect. 87 and 217 kDa PEI rendered the highest transfection rates in HEK 293 and Hep G2 cell lines (>50% transfected cells) with minimal cell toxicity. In conclusion, our results indicate that fully deacylated PEI of 87 and 217 kDa are useful DNA vehicles for non-viral transfection of primary cultures of bovine fetal fibroblast and HEK 293 and Hep G2 cell lines.     

Strategy for increased efficiency of transfection in human cell lines using radio frequency electroporation

Traditional electroporation devices use direct current electric fields to stimulate the uptake of oligonucleotides, plasmids, short peptides, and proteins into a variety of cell types. A variation of this widely used technique is now available which relies on radio frequency (RF) electrical pulses. This oscillating type of electrical field reportedly elicits greater uptake of plasmid DNA across the plasma membrane. We evaluated a protocol for RF electroporation of the a human embryonic kidney cell line and a Burkitt's lymphoma (BL) cell line for effeciency of transfection by RF electroporation. The plasmid EGFP, which codes for the widely used fusion protein, enhanced green fluorescent protein (EGFP), was used as a reporter of plasmid uptake after transfections. Transfection efficiency consistently increased approximately 30% from that typically obtained with conventional DC type electroporation and was accompanied by greater survivability of cells. Additionally, in some instances, percent transfection efficiency increased to over 70%. Thus, RF electroporation represents an improved methodology for transfection of human cell lines. Moreover, the RF protocol is simple to incorporate in laboratories already utilizing conventional electroporation devices and techniques.     

Transmembrane routes of cationic liposome-mediated gene delivery using human throat epidermis cancer cells

For studying the mechanism of cationic liposome-mediated transmembrane routes for gene delivery, various inhibitors of endocytosis were used to treat human throat epidermis cancer cells, Hep-2, before transfection with Lipofectamine 2000/pGFP-N2 or Lipofectamine 2000/pGL3. To eliminate the effect of inhibitor toxicity on transfection, the RLU/survival rate was used to represent the transfection efficiency. Chlorpromazine and wortmannin, clathrin inhibitors, decreased transfection efficiency by 44 % (100 μM) and 31 % (100 nM), respectively. At the same time, genistein, a caveolin inhibitor, decreased it by 30 % (200 μM). Thus combined transmembrane routes through the clathrin and caveolae-mediated pathways were major mechanisms of cell uptake for the cationic liposome-mediated gene delivery. After entering the cells, microtubules played an important role on gene delivery as vinblastine, a microtubulin inhibitor, could reduce transfection efficiency by 41 % (200 nM).     

Successful genetic transduction in vivo into synovium by means of electroporation

This present study aims at establishing a novel in vivo gene delivery system for intra-articular tissues. Plasmid DNA (pDNA) carrying the firefly luciferase or enhanced green fluorescent protein (EGFP) genes as markers was injected into a joint space and electric stimuli were given percutaneously with a pair of electrodes. Injection with naked pDNA alone did not induce any detectable level of luciferase activity, whereas electroporation at 25-500 V/0.7 cm resulted in a significant expression of the marker gene in the synovium. The expression level depended on the voltage, the optimum transfection being achieved at 150 V/0.7 cm. When the Epstein-Barr virus (EBV)-based plasmid vectors harboring the EBV nuclear antigen 1 (EBNA1) gene and oriP sequence were substituted for conventional pDNA, the transfection efficiency was increased approximately 5-10 times. Histological examination of the EGFP gene-transfected joints revealed that the marker gene was expressed in the synovial membrane while other intra-articular tissues such as articular cartilage were negative for the transgene product. Transgene-specific mRNA was demonstrated in synovium but not in other organs as estimated by RT-PCR analysis. The present results strongly suggest that in vivo electroporation is a quite simple, safe, and effective gene delivery method that could be applicable to gene therapy against articular diseases.     

An optimized electroporation protocol for transfection of sensitive cell lines using basic laboratory equipment

Using previously reported protocols, electroporation of 21 very sensitive human cell lines showed poor results with high mortality and low transfection efficiency. Therefore, the influence of several electroporation parameters on transfection success was analyzed. The adjustment of the time constant proved to be most important for optimization of transfection results. Time constant was modulated by changing medium resistance via volume or ionic strength, yielding an average transfection efficiency of 25% and mortality rates below 60%.     

Transient transfection of purified Babesia bovis merozoites

Transient transfection of intraerythrocytic Babesia bovis parasites has been previously reported. In this study, we describe the development and optimization of methods for transfection of purified B. bovis merozoites using either nucleofection (Amaxa) or conventional electroporation (Gene Pulser II, BioRad). Initially, the optimal buffer ("Plasmodium 88A6") and program (v-24) for nucleofection of free merozoites with a plasmid containing the luciferase gene as a reporter were determined. Using the same reporter plasmid, optimal voltage, capacitance and resistance for transfecting free merozoites by electroporation were defined to be 1.2 kV/25 microF/200 Omega. Using these optimal parameters, analysis of the time course of luciferase expression using either system to transfect free B. bovis merozoites showed high enzyme activity at 24h, with a rapid decline thereafter. Nucleofection was approximately five times more effective than electroporation when using a small quantity (2 microg) of DNA, while electroporation was twice as effective as nucleofection when a larger quantity of plasmid DNA (100 microg) was used. Parasite viability was significantly higher when using nucleofection when compared to electroporation regardless of the amount of DNA used. Comparison of luciferase expression after transfection of merozoites with circular, linearized, or double digested plasmid indicated that intact, circular plasmid was necessary for optimal luciferase expression. Overall, the results provide a basis for optimal transfection of purified B. bovis merozoites using either nucleofection or conventional electroporation. However, nucleofection is significantly more efficient when transfecting either circular or restriction digested DNA in the 2-10 microg range.     

Electroporation-mediated transient gene expression in isolated scutella of Hordeum vulgare

Electroporation was used to introduce DNA into excised scutella of immature embryos of Hordeum vulgare L. cvs Golden Promise and Delita. Using the firefly luciferase gene as reporter, parameters were analyzed for high transient gene expression while maintaining tissue viability. Enzymatic wounding was necessary for DNA uptake. The optimized protocol involves use of linearized DNA and addition of 15% (w/v) polyethylene glycol at a field strength of 950 V cm⁻¹ and approximately 56 ms pulse length. A one-day preculture was required for obtaining callus after electroporation. Transient gene expression was further demonstrated using the β-glucuronidase gene. Blue spots were detected at the abaxial scutellar surface, indicating that cells competent for somatic embryogenesis are also amenable to transfection by electroporation.     

Dependence of the efficacy of transfecting muscle fibers in vivo on electroporation conditions

This study is a survey of in vivo experiments on transfection of laboratory mouse muscle fibers by electroporation using an original device generating electric impulses. Transfection efficiency proved to depend on DNA dose and the number of electric impulses. It can be increased significantly by electroporation at varying pulse burst polarity. At both direct electrode application to muscles and electroporation through the skin, the muscle fiber transfection was more efficient under electroporation conditions much milder than those usually reported. The use of electroporation method for gene therapy of Duchenne muscular dystrophy is discussed.     

Efficient in vivo gene delivery using modified Tat peptide with cationic lipids

A combination of modified HIV-1 Tat (mTat) peptide and cationic lipids, FuGENE HD (FH), dramatically enhanced transfection efficiency across a range of cell lines when compared to mTat or FH alone (Biomaterials 35:1705–1715 2014). The efficiency of this Tat peptide combination was significantly higher than many commercial non-viral vectors. In this present study, we tested the feasibility of this non-viral vector, mTat/FH, in vivo using plasmid DNA encoding a luciferase gene. The results of the in vivo studies showed that animals administered mTat/FH/DNA intramuscularly had significantly higher and longer luciferase expression (≈7 months) than those with mTat/DNA, FH/DNA, or DNA alone. Histological evaluation showed little immune response in the muscles, livers, and kidneys of mice administered with the mTat/FH. The combination of mTat with FH could significantly improve transfection efficiency, expanding the potential use of non-viral gene vectors in vivo.     

Influence of Electroporation Conditions on Transfection of Muscle Fibers in Vivo

This study is a survey of in vivo experiments on transfection of laboratory mouse muscle fibers by electoporation using an original device generating electric impulses. Transfection efficiency proved to depend on DNA dose and the number of electric impulses. It can be increased significantly by electroporation at varying pulse burst polarity. At both direct electrode application to muscles and electroporation through the skin, the muscle fiber transfection was more efficient under electroporation conditions much milder than those usually reported. The use of electroporation method for gene therapy of Duchenne muscular dystrophy is discussed.     

Comparison of non-viral transfection methods in melanoma cell primary cultures

Melanoma primary cultures were transiently transfected via electroporation and lipofection for comparison. Transfection efficiency was superior with electroporation (58+/-9%) as compared to lipofection (23+/-9%) as determined by enhanced green fluorescent plasmid (EGFP) transfection. Secretion of IL-2 persisted for up to 3 weeks after electroporation. The increase in sensitivity against immunologic effector cells by transfection with IL-2 was not significant. Our results show the feasibility of a gene transfer into primary human melanoma cells, different from retroviral transduction.     

A novel cell transfection platform based on laser optoporation mediated by Au nanostar layers

The recently developed laser‐induced cell transfection mediated by Au nanoparticles is a promising alternative to the well‐established lipid‐based transfection or to electroporation. Optoporation is based on the laser plasmonic heating of nanoparticles located near the cell membrane. However, the uncontrollable cell damage from intense laser pulses and from random attachment of nanoparticles may be crucial for transfection. We present a novel plasmonic optoporation technique that uses Au nanostar layers immobilized in culture microplate wells. HeLa cells were grown directly on Au nanostar layers, after which they were subjected to continuous‐wave 808 nm laser irradiation. An Au monolayer density ~15 μg/cm² and an absorbed energy of about 15 to 30 J were found to be optimal for optoporation. Propidium iodide molecules were used as model penetrating agent. The transfection efficiency evaluated using fluorescence microscopy for HeLa cells transfected with pGFP under optimized optoporation conditions (95% ± 5%) was similar to the efficiency of TurboFect. The technique's efficiency (295 ± 10 relative light units, RLU), demonstrated by transfecting HeLa cells with the pCMV‐GLuc 2 control plasmid, was greater than that obtained by transfection of HeLa cells with the TurboFect agent (220 ± 10 RLU). The cell viability in plasmonic optoporation (92% ± 7%), too, was greater than that in transfection with TurboFect (75% ± 7%).      

DNA Transfection of Bone Marrow Stromal Cells Using Microbubble-Mediated Ultrasound and Polyethylenimine: An In Vitro Study

Non-viral vector transfection efficiency is an issue affecting the clinical application of stem cell gene therapy. This study makes use of the synergistic effect of combining ultrasound (US) with microbubbles (MB) and polyethylenimine (PEI) to increase DNA transfection efficiency, which will enhance the efficiency of gene transfer to bone marrow stromal cells (BMSCs). The optimal parameters for primary-cultured rat-BMSC DNA transfection were examined. The study was arranged based on uniform design. Using a construct containing hepatocyte growth factor (HGF) tagged with enhanced green fluorescent protein (pEGFP-HGF) as example, the mixture of BMSCs, MB, and PEI:DNA complex were exposed to US with frequency of 1 MHz and 10 % duty cycle pulses. Other factors such as acoustic intensity (Q), MB dosage, and total treatment time (T) were also tested. The results were analyzed by regression analysis. Using the best match of parameters, Q = 0.6 W/cm², MB = 10⁶/ml, T = 30 s, different groups were compared. The cooperativity of MB-mediated US and PEI enhanced the gene transfection efficiency by nearly 38-times compared to the DNA without US group. Furthermore, the expression of HGF protein was confirmed by Western blot. The eGFP could be not only seen mainly at the cytoplasm, but also seen in the nucleus in a small proportion of the cells (<10 %) for up to 7 observed days. The transfected BMSCs maintained their capability of multi-directional differentiation and reproductive activity. Our results provide useful information in establishing a novel non-viral transfection method, which may be applied to clinical application in stem cell gene therapy.     

Properties of electroporation-mediated DNA transfer in Escherichia coli

Efficient and reproducible DNA-transfection was attained in E. coli, by electroporation. The yield of the transfectants was affected by pretreatment of the recipient cells as well as by the composition of the electroporation medium. Using a single pulse procedure, relationships among the electrical parameters, the transfection efficiency, and the cellular viability were investigated in 10 mM Tris-HCl buffer (pH 7.5) containing 5% sucrose. Certain sodium salts (e.g., citrate, phosphate, and sulfate) were promotive, whereas Mg2+, DEAE-dextran, and polyvinylpyrrolidone were inhibitory to the transfection. Heterologous nucleic acids (native DNA, denatured DNA, and tRNA) exerted only a marginal effect on transfection with a viral replicative-form DNA. The efficiency of DNA transfer was affected by culture conditions, and bacteria grown at a higher temperature were more competent. The electroporation system was more efficient than an improved CaCl2 method, not only in transfection with viral single- and double-stranded DNAs, but also in transformation with plasmid DNAs.