Effects of electroporation pulse wave on the incorporation of viral RNA into tobacco protoplasts

The uptake and expression of cucumber mosaic viral (CMV) RNA by tobacco protoplasts was examined using both square wave and exponential wave electroporation pulses. These electropulses, when supplied at sufficient field strength for a critical duration, enabled RNA to be incorporated and expressed in more than 60% of the surviving protoplasts. The results of experiments using these two electroporation wave forms showed significant differences in RNA uptake and expression. The number of viable protoplasts and cells showing expression of RNA was higher over a much broader range of experimental conditions using the square rather than exponential wave generator, even when both machines were optimized for maximal performance. However, at a narrow range of low field strengths the exponential wave pulse generated a higher percentage of transformants than did the square wave pulse. It was shown that after an electroporation pulse from either wave form, there were viable cells which expressed foreign RNA at predictable levels.     

DNA transfer into vascular smooth muscle using fusigenic Sendai virus (HJV)-liposomes

Manipulation of the genetic machinery of cells both in vitro and in vivo is becoming an ever more important means of elucidating pathways of molecular and cellular biochemistry. In addition, gene therapy has been proposed as a novel and potentially powerful treatment for both inherited and acquired diseases. Successful gene transfer and gene blockade generally depend on high efficiency delivery of exogenous DNA or RNA into living cells, and much effort has therefore been focused on the development of methods for achieving this delivery in a safe and effective manner. We describe here our application of fusigenic Sendai virus (HVJ)-liposome technology toward the effective delivery of DNA into vascular smooth muscle cells (VSMC) in cell culture. Cellular uptake and intracellular distribution of oligodeoxynucleotide (ODN) after transfection with HVJ-liposome complexes was characterized using fluorescent (FITC)-labeled ODN, and the biologic effect of HVJ-liposome mediated transfection was demonstrated via inhibition of DNA synthesis in cultured VSMC using antisense ODN against basic fibroblast growth factor.     

Effects of pulse length and pulse strength on transfection by electroporation

The relative importance of pulse field strength E and pulse length tau 1/2 (half decay time of an exponential decay pulse) on the stable transfection frequency for HeLa or HUT-78 cells was investigated. Cells were transfected with plasmids containing the promoter and drug resistant genes pRSVgpt or pRSVneo by electroporation. The stable transfection frequency was assayed using the marker rescue technique. The transfection frequency increases with increasing values of E tau 1/2. For a given pulse length, the transfection frequency is proportional to the power of the pulse (E2 tau 1/2). Pulses with half decay times of 2.2 to 4.6 ms appear to be more efficient than 0.275 to 0.31 ms for stable transfection of HeLa cells.     

Electroporation enhances c-myc antisense oligodeoxynucleotide efficacy.

Obtaining high transfection efficiencies and achieving appropriate intracellular concentrations and localization are two of the most important barriers to the implementation of gene targeted therapy. The efficiency of endogenous uptake of oligodeoxynucleotides (ODNs) varies from cell type to cell type and may be a limiting factor of antisense efficacy. The use of electroporation to obtain high intracellular concentrations of a synthetic ODN in essentially 100% of viable cells is described. It is also shown that the transfected ODNs initially localize to the nucleus and remain there for at least 48 hours. The cellular trafficking of electroporated ODNs is shown to be an energy dependent process. Targeting of the c-myc proto-oncogene of U937 cells by electroporation of phosphorothioate-modified ODNs results in rapid and specific suppression of this gene at ODN concentrations much lower than would otherwise be required. This technique appears to be applicable to a variety of cell types and may represent a powerful new investigate tool as well as a promising approach to the ex vivo treatment of hematologic disorders.     

Electroporation Facilitates Introduction of Reporter Transgenes and Virions into Schistosome Eggs

Background

The schistosome egg represents an attractive developmental stage at which to target transgenes because of the high ratio of germ to somatic cells, because the transgene might be propagated and amplified by infecting snails with the miracidia hatched from treated eggs, and because eggs can be readily obtained from experimentally infected rodents.

Methods/Findings

We investigated the utility of square wave electroporation to deliver transgenes and other macromolecules including fluorescent (Cy3) short interference (si) RNA molecules, messenger RNAs, and virions into eggs of Schistosoma mansoni. First, eggs were incubated in Cy3-labeled siRNA with and without square wave electroporation. Cy3-signals were detected by fluorescence microscopy in eggs and miracidia hatched from treated eggs. Second, electroporation was employed to introduce mRNA encoding firefly luciferase into eggs. Luciferase activity was detected three hours later, whereas luciferase was not evident in eggs soaked in the mRNA. Third, schistosome eggs were exposed to Moloney murine leukemia virus virions (MLV) pseudotyped with vesicular stomatitis virus glycoprotein (VSVG). Proviral transgenes were detected by PCR in genomic DNA from miracidia hatched from virion-exposed eggs, indicating the presence of transgenes in larval schistosomes that had been either soaked or electroporated. However, quantitative PCR (qPCR) analysis determined that electroporation of virions resulted in 2–3 times as many copies of provirus in these schistosomes compared to soaking alone. In addition, relative qPCR indicated a copy number for the proviral luciferase transgene of ∼20 copies for 100 copies of a representative single copy endogenous gene (encoding cathepsin D).

Conclusions

Square wave electroporation facilitates introduction of transgenes into the schistosome egg. Electroporation was more effective for the transduction of eggs with pseudotyped MLV than simply soaking the eggs in virions. These findings underscore the potential of targeting the schistosome egg for germ line transgenesis.     

Optimisation of transfection conditions of CD34+ hematopoietic cells derived from human umbilical cord blood

Human umbilical cord blood is frequently used as a source of transplantable hematopoietic cells and more recently as a target of gene therapy - a new approach for treatment of various disorders. The aim of our study was optimisation of the transfection conditions of cord blood-derived CD34(+) hematopoietic cells. Mononuclear cells fraction was isolated from cord blood samples by density gradient centrifugation. Subsequently, CD34(+) hematopoietic cells were separated on immunomagnetic MiniMACS columns. Pure population of CD34(+) cells was incubated in a serum free medium supplemented with thrombopoietin, stem cell factor and Flt-3 ligand for 48 h and then transfected with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene. We studied the influence of various pulse settings and DNA concentrations on the transfection efficiency, measured by flow cytometry as the fluorescence of target cells due to the expression of EGFP. The optimal settings were as follows: 4 mm cuvette, 1600 microF, 550 V/cm, and 10 microg of DNA per 500 microl. With these settings we obtained a high transfection frequency (41.2%) without a marked decrease of cell viability. An increase of the pulse capacitance and/or of DNA concentration resulted in a greater electroporation efficiency, but also in a decrease of cell viability. In conclusion, the results described here allow one to recommend electroporation as an efficient method of gene delivery into CD34(+) hematopoietic cells derived from human umbilical cord blood.     

High efficiency,Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes^1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies^9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies¹³. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells^14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies^{19, 24-26}. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection²⁷. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures^17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.     

Transfection of human monocyte-derived dendritic cells with CpG oligonucleotides

Monocyte-derived dendritic cells (mDC), the most frequently applied DC subset in clinical studies, which can be obtained easily from peripheral blood monocytes after incubation with GM-CSF and IL-4, have not been clearly demonstrated to be activated by CpG oligodeoxynucleotides (ODN). The development of novel molecular strategies - such as the use of CpG-ODN - to increase immunological functions and thus improve the therapeutic efficiency of mDC vaccines in the treatment of malignant diseases is highly desirable. CpG-ODN need to be internalized into specific intracellular compartments to be active. Therefore, we applied electroporation and lipofection and compared these techniques with incubation to overcome possible defects in localization. Conditions of CpG-ODN transfection of these cells were optimized using fluorescein-marked ODN 2216. We were able to achieve high transfection efficiencies with various methods of delivery. However, we did not observe increased expression of maturation-associated and functionally relevant surface antigens (CD14, HLA-DR, CD40, CD83, CD80 and CD86), significant secretion of IL-12 and IFN-alpha in culture supernatant, or enhanced antitumour activation of cytokine-induced killer cells. In conclusion, our results show that non-viral transfection of CpG-ODN is not sufficient to overcome resistance of mDC to CpG activation.     

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.     

Electroporated intact BY-2 tobacco culture cells as a model of transient expression study

Transfer of foreign genes into plant cells can be accomplished by several methods: agrobacterium-mediated, microinjection, biolistic particle bombardment and electroporation. The last one is frequently used for transfection of plant protoplasts for transient gene expression. Electroporation is a simple procedure and allows transfecting a large number of cells at one time. Square wave-modulated porators are the most efficient for introducing expression cassettes into plant protoplasts. Based on a protocol developed by Wu & Feng (Plant Cell Reports, 1999, 18, 381-386), we optimized conditions for transfection of intact Nicotiana tabacum BY-2 cells using square wave-modulated electroporator. To simplify screening for transfected gene expression we used constructs with a GFP marker gene.     

Nanostructure of polyplexes formed between cationic diblock copolymer and antisense oligodeoxynucleotide and its influence on cell transfection efficiency

Although various cationic polymers have been used to condense anionically charged DNA to improve their transfection efficiency, there is still a lack of fundamental understanding about how to control the nanostructure and charge of the polyplexes formed and how to relate such information to cell transfection efficiency. In this work, we have synthesized a weak cationic and phosphorylcholine-containing diblock copolymer and used it as a model vector to deliver an antisense oligodeoxynucleotide (ODN) into HeLa cells. Small angle neutron scattering (SANS) was used to determine the copolymer/ODN polyplex structure. The SANS data revealed the formation of polyplex nanocylinders at high copolymer (N)/ODN (P) charge ratios, where N symbolizes the amine groups on the copolymer and P symbolizes the phosphate groups. However, the cylindrical lengths remained constant, indicating that the ODN binding over this region did not alter the cylindrical shape of the copolymer in solution. As the N/P ratio decreased and became close to unity the polyplex diameters remained constant, but their lengths increased substantially, suggesting the end-to-end bridging by ODN binding between copolymer cylinders. As the N/P ratios went below unity (with ODN in excess), the polyplex diameters increased substantially, indicating different ODN bridging to bundle the small polyplexes together. Transfection studies from HeLa cells indicated a steady increase in transfection efficiency with increasing cationic charge and decreasing polyplex size. Cell growth inhibition assay showed significant growth inhibition by the polyplexes coupled with weak cytotoxicity, indicating effective ODN delivery. While this study has confirmed the overall charge effect, it has also revealed progressive structural changes of the polyplexes against varying charge ratio, thereby providing useful insight into the mechanistic process behind the ODN delivery.     

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.     

Enhanced Nanomagnetic Gene Transfection of Human Prenatal Cardiac Progenitor Cells and Adult Cardiomyocytes

Magnetic nanoparticle-based gene transfection has been shown to be an effective, non-viral technique for delivery of both plasmid DNA and siRNA into cells in culture. It has several advantages over other non-viral delivery techniques, such as short transfection times and high cell viability. These advantages have been demonstrated in a number of primary cells and cell lines. Here we report that oscillating magnet array-based nanomagnetic transfection significantly improves transfection efficiency in both human prenatal cardiac progenitor cells and adult cardiomyocytes when compared to static magnetofection, cationic lipid reagents and electroporation, while maintaining high cell viability. In addition, transfection of adult cardiomyocytes was improved further by seeding the cells onto Collagen I-coated plates, with transfection efficiencies of up to 49% compared to 24% with lipid reagents and 19% with electroporation. These results demonstrate that oscillating nanomagnetic transfection far outperforms other non-viral transfection techniques in these important cells.     

Optimized conditions for transgenesis of the ascidian Ciona using square wave electroporation

The protochordate Ciona has recently become an important model organism for the study of developmental gene regulation, in part because transient transgenic embryos can be produced rapidly and reliably using electroporation. Published methods are currently for the use of electroporation devices delivering exponentially decaying pulses. However, some workers have advocated the use of square wave electroporation devices for eukaryotic transgenics. This paper presents results of experiments to find optimal conditions for the use of square-wave electroporation in the ascidian Ciona. In the present analysis, a single pulse of 90 msec duration at 63–75 V/cm was found to give an optimal balance of a high proportion of cells transformed with the transgene, and a low level of abnormal development. Forty micrograms of DNA per electroporation is sufficient for effective visualization of reporter gene expression, although doses up to 100 μg provide higher proportions of transformed cells. In side-by-side comparison with exponential pulse electroporation, square wave pulses give similar penetrance of transgene expression, along with lower proportions of embryos with abnormal development at higher amounts of input DNA.     

Novel cationic lipophilic peptides for oligodeoxynucleotide delivery

In search of new oligodeoxynucleotide (ODN) delivery agents, we evaluated novel peptides derived from core peptide H-GLRILLLKV-OH (CP). CP is a fragment designed from the T-cell antigen receptor (TCR) alpha-chain transmembrane sequence. CP was able to enter cells including T-cells and inhibited interleukin-2 (IL-2) production. To examine the effect of increased lipophilicity on cellular uptake and activity of CP, a lipoamino acid (2-aminododecanoic acid) was incorporated into peptide CP resulting in 2-aminodecanoyl-CP (LP). The toxicity of CP and LP was assessed by measuring the haemolytic activity. Neither compound caused any haemolysis of red blood cells. We have also compared the biological activities of the CP and LP. Using a T-cell antigen presentation assay, the more lipophilic LP caused greater inhibition of IL-2 production than the parent CP in the antigen stimulated T-cells. The LP also showed increased permeability than CP in the Caco-2 cell assay. We utilised the enhanced cell permeability property of LP in oligodeoxynucleotide ODN1 delivery. Isothermal titration calorimetry (ITC) suggested that CP and LP complex with ODN1 in a 12:1 (CP:ODN1) and 15:1 (LP:ODN1) ratio. These complexes were then transfected into human retinal pigment epithelial cells. The level of transfection was measured by the decreased production of the protein human vascular endothelial growth factor (hVEGF). The results revealed greater transfection efficiency for both CP and LP (47%, 55% more inhibition) compared to commercially available transfection agent cytofectin GSV. These results suggested that the CP and particularly its lipophilic analogue LP have the potential to be used as oligodeoxynucleotide delivery systems.     

Optimizing electroporation parameters for a variety of human hematopoietic cell lines

The parameters affecting electroporation of four human hematopoietic cell lines were investigated. The optimal conditions for electroporation are described for both transient and stable expression of foreign genes. A correlation exists between the levels of transient gene expression and stable transfection frequency. In addition, linear DNA yields higher stable transfection frequencies than supercoiled DNA. The cumulative results indicate that electroporation is a simple and useful method for obtaining transient and stable expression of foreign genes in human hematopoietic cells.     

Rapid optimization of electroporation conditions for plant cells,protoplasts, and pollen

The optimization of electroporation conditions for maximal uptake of DNA during direct gene transfer experiments is critical to achieve high levels of gene expression in transformed plant cells. Two stains, trypan blue and fluorescein diacetate, have been applied to optimize electroporation conditions for three plant cell types, using different square wave and exponential wave electroporation devices. The different cell types included protoplasts from tobacco, a stable mixotrophic suspension cell culture from soybean with intact cell walls, and germinating pollen from alfalfa and tobacco. Successful electroporation of each of these cell types was obtained, even in the presence of an intact cell wall when conditions were optimized for the electroporation pulse. The optimal field strength for each of these cells differs, protoplasts having the lowest optimal pulse field strength, followed by suspension cells and finally germinating pollen requiring the strongest electroporation pulse. A rapid procedure is described for optimizing electroporation parameters using different types of cells from different plant sources.     

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.