Showing papers on "Electroporation published in 2007"
TL;DR: There appears to be complete ablation to the margin of blood vessels without compromising the functionality of the blood vessels, which suggests that IRE is a promising method for treatment of tumors near blood vessels (a significant challenge with current ablation methods).
Abstract: Irreversible electroporation (IRE) is a new tissue ablation technique in which micro to millisecond electrical pulses are delivered to undesirable tissue to produce cell necrosis through irreversible cell membrane permeabilization. IRE affects only the cell membrane and no other structure in the tissue. The goal of the study is to test our IRE tissue ablation methodology in the pig liver, provide first experience results on long term histopathology of IRE ablated tissue, and discuss the clinical implications of the findings. The study consists of: a) designing an IRE ablation protocol through a mathematical analysis of the electrical field during electroporation; b) using ultrasound to position the electroporation electrodes in the predetermined locations and subsequently to monitor the process; c) applying the predetermined electrotroporation pulses; d) performing histolopathology on the treated samples for up to two weeks after the procedure; and e) correlating the mathematical analysis, ultrasound data...
681 citations
TL;DR: Conditional regulation of gene expression from electroporated plasmids in the postnatal rat retina and the embryonic mouse brain is demonstrated.
Abstract: In vivo electroporation is a powerful technique for the introduction of genes into organisms. Temporal and spatial regulation of expression of introduced genes, or of RNAi, would further enhance the utility of this method. Here we demonstrate conditional regulation of gene expression from electroporated plasmids in the postnatal rat retina and the embryonic mouse brain. For temporal regulation, Cre/loxP-mediated inducible expression vectors were used in combination with a vector expressing a conditionally active form of Cre recombinase, which is activated by 4-hydroxytamoxifen. Onset of gene expression was regulated by the timing of 4-hydroxytamoxifen administration. For spatial regulation, transgenes were expressed by using promoters specific for rod photoreceptors, bipolar cells, amacrine cells, Muller glia or progenitor cells. Combinations of these constructs will facilitate a variety of experiments, including cell-type-specific gene misexpression, conditional RNAi, and fate mapping of progenitor and precursor cells.
613 citations
TL;DR: The first successful use of irreversible electroporation for the minimally invasive treatment of aggressive cutaneous tumors implanted in mice was reported, and induced complete regression in 12 out of 13 treated tumors, in the absence of tissue heating.
Abstract: We report the first successful use of irreversible electroporation for the minimally invasive treatment of aggressive cutaneous tumors implanted in mice. Irreversible electroporation is a newly developed non-thermal tissue ablation technique in which certain short duration electrical fields are used to permanently permeabilize the cell membrane, presumably through the formation of nanoscale defects in the cell membrane. Mathematical models of the electrical and thermal fields that develop during the application of the pulses were used to design an efficient treatment protocol with minimal heating of the tissue. Tumor regression was confirmed by histological studies which also revealed that it occurred as a direct result of irreversible cell membrane permeabilization. Parametric studies show that the successful outcome of the procedure is related to the applied electric field strength, the total pulse duration as well as the temporal mode of delivery of the pulses. Our best results were obtained using plate electrodes to deliver across the tumor 80 pulses of 100 µs at 0.3 Hz with an electrical field magnitude of 2500 V/cm. These conditions induced complete regression in 12 out of 13 treated tumors, (92%), in the absence of tissue heating. Irreversible electroporation is thus a new effective modality for non-thermal tumor ablation.
481 citations
TL;DR: A transposon-mediated gene transfer is developed, a novel technique in chicken manipulations that facilitates an integration of a transgene into the genome when co-acting with Tol2 transposase and could be experimentally induced upon tetracycline administration at relatively late stages such as E6, where a variety of organogenesis are underway.
253 citations
TL;DR: This protocol details an in vivo transient expression system to study the subcellular localization and dynamic associations of plant proteins using protoplasts freshly prepared from Arabidopsis or tobacco BY-2 suspension cultured cells.
Abstract: Transient expression of fluorescent fusion proteins in plant cells has dramatically facilitated our study of newly identified genes and proteins. This protocol details an in vivo transient expression system to study the subcellular localization and dynamic associations of plant proteins using protoplasts freshly prepared from Arabidopsis or tobacco BY-2 suspension cultured cells. The method relies on the transformation of DNA constructs into protoplasts via electroporation. The whole protocol is comprised of three major stages: protoplast generation and purification, transformation of DNA into protoplasts via electroporation and incubation of protoplasts for protein analysis. Similar to stably transformed cell lines, transformed protoplasts are compatible with protein localization studies, pharmaceutical drug treatment and western blot analysis. This protocol can be completed within 11-24 h from protoplast production to protein detection.
210 citations
TL;DR: The overall long term pattern of change in conductivity after electroporation is complex and different between reversible and irreversible groups, which suggests the superposition of different phenomena which together affect the electrical properties.
176 citations
TL;DR: A method for the genetic modification of HESCs using electroporation, the preferred method for introduction of DNA into cells in which the desired outcome is gene targeting is described.
Abstract: The ability to genetically modify human embryonic stem cells (HESCs) will be critical for their widespread use as a tool for understanding fundamental aspects of human biology and pathology and for their development as a platform for pharmaceutical discovery. Here, we describe a method for the genetic modification of HESCs using electroporation, the preferred method for introduction of DNA into cells in which the desired outcome is gene targeting. This report provides methods for cell amplification, electroporation, colony selection and screening. The protocol we describe has been tested on four different HESC lines, and takes approximately 4 weeks from electroporation to PCR screening of G418-resistant clones.
166 citations
TL;DR: The possibility to monitor in vivo cell electroporation during pulse delivery, and to adjust the electric field strength on real time, within a few microseconds after the beginning of the pulse, is reported to ensure efficacy and safety of the procedure.
147 citations
TL;DR: The combined effects mediated by in vivo EP serves as a potent adjuvant for the NS3/4A-based DNA vaccine and enhances protein expression and inflammation at the injection site following in vivoEP contributed to the priming of in vivo functional immune responses.
Abstract: The mechanisms by which in vivo electroporation (EP) improves the potency of i.m. DNA vaccination were characterized by using the hepatitis C virus nonstructural (NS) 3/4A gene. Following a standard i.m. injection of DNA with or without in vivo EP, plasmid levels peaked immediately at the site of injection and decreased by 4 logs the first week. In vivo EP did not promote plasmid persistence and, depending on the dose, the plasmid was cleared or almost cleared after 60 days. In vivo imaging and immunohistochemistry revealed that protein expression was restricted to the injection site despite the detection of significant levels of plasmid in adjacent muscle groups. In vivo EP increased and prolonged NS3/4A protein expression levels as well as an increased infiltration of CD3+ T cells at the injection site. These factors most likely additively contributed to the enhanced and broadened priming of NS3/4A-specific Abs, CD4+ T cells, CD8+ T cells, and gamma-IFN production. The primed CD8+ responses were functional in vivo, resulting in elimination of hepatitis C virus NS3/4A-expressing liver cells in transiently transgenic mice. Collectively, the enhanced protein expression and inflammation at the injection site following in vivo EP contributed to the priming of in vivo functional immune responses. These localized effects most likely help to insure that the strength and duration of the responses are maintained when the vaccine is tested in larger animals, including rabbits and humans. Thus, the combined effects mediated by in vivo EP serves as a potent adjuvant for the NS3/4A-based DNA vaccine.
144 citations
TL;DR: It is concluded that electroporation is a simple approach that permits Ca2+ indicator loading of multiple cells with low background staining within a short amount of time, which makes it especially well suited for functional imaging of subcellular Ca 2+ dynamics in small neuronal networks.
Abstract: Studies of subcellular Ca(2+) signaling rely on methods for labeling cells with fluorescent Ca(2+) indicator dyes. In this study, we demonstrate the use of single-cell electroporation for Ca(2+) indicator loading of individual neurons and small neuronal networks in rat neocortex in vitro and in vivo. Brief voltage pulses were delivered through glass pipettes positioned close to target cells. This approach resulted in reliable and rapid (within seconds) loading of somata and subsequent complete labeling of dendritic and axonal arborizations. By using simultaneous whole-cell recordings in brain slices, we directly addressed the effect of electroporation on neurons. Cell viability was high (about 85%) with recovery from the membrane permeabilization occurring within a minute. Electrical properties of recovered cells were indistinguishable before and after electroporation. In addition, Ca(2+) transients with normal appearance could be evoked in dendrites, spines, and axonal boutons of electroporated cells. Using negative-stains of somata, targeted single-cell electroporation was equally applicable in vivo. We conclude that electroporation is a simple approach that permits Ca(2+) indicator loading of multiple cells with low background staining within a short amount of time, which makes it especially well suited for functional imaging of subcellular Ca(2+) dynamics in small neuronal networks.
131 citations
TL;DR: Using the recently developed 96-well shuttle system, reproducible high-throughput expression of various transgenes is now feasible on primary neurons, for example large-scale RNAi analyses to downregulate gene expression.
Abstract: Transfection of foreign DNA is widely used to study gene function. However, despite the development of numerous methods, the transfer of DNA into postmitotic cells, such as neurons, remains unsatisfactory with regard to either transfection efficiency or cytotoxicity. Nucleofection overcomes these limitations. Direct electroporation of expression plasmids or oligonucleotides into the nucleus ensures both good cell viability and consistently high transfection rates. This allows biochemical analyses of transfected neurons, for example, western blot analyses of protein levels after RNA interference (RNAi) knockdown or microRNA transfection. We provide comprehensive protocols for performing nucleofection with high efficiency on primary neurons. The focus is on the recently developed 96-well shuttle system, which allows the simultaneous testing of up to 96 different plasmids or experimental conditions. Using this system, reproducible high-throughput expression of various transgenes is now feasible on primary neurons, for example large-scale RNAi analyses to downregulate gene expression. The protocol typically takes between 2 and 3 h.
TL;DR: It was observed that using this combination method, 100% human LoVo cancer cells were photokilled within 90 min, while only 39% of the normal cells were killed under the irradiation of the ultraviolet (UV) light (365 nm).
TL;DR: The averaged tissue conductivity in both models increases during IRE pulses because of electroporation, which leads to greater electrical dissipation (heating) and, thus, to larger temperature increases than suggested by tissue models with passive and static electrical properties.
Abstract: Local and drug-free tissue treatment by irreversible electroporation (IRE) involves the creation of aqueous pores in a cell's plasma membrane (PM) and leads to non-thermal cell death by necrosis. To investigate explicit pore-based effects we use two-dimensional system models with different spatial scales. The first is a multicellular system model (spatial scale 100 mum) that has irregularly shaped cells, and quantitatively describes dynamic (creation and destruction, evolution in pore size) pore behavior at the PM. The second is a tissue model (spatial scale 200 mm) that is constructed from a unit cell and uses the asymptotic (fixed pore size) electroporation model. Both system models show that significant redistribution of fields and currents occurs through transient PM pores. Pore histograms for the multicellular model demonstrate the simultaneous presence of small and large pores during IRE pulses. The associated significant increase of PM permeability may prove to be essential to understanding how cell death by necrosis occurs. The averaged tissue conductivity in both models increases during IRE pulses because of electroporation. This leads to greater electrical dissipation (heating) and, thus, to larger temperature increases than suggested by tissue models with passive and static electrical properties.
TL;DR: This system, which mounts on an inverted microscope, obviates many issues typically associated with prototypical microfluidic chip setups and, more importantly, offers well-controlled and reproducible parallel pressure and electrical application to individual cells for repeatability.
Abstract: Rapid well-controlled intracellular delivery of drug compounds, RNA, or DNA into a cell – without permanent damage to the cell – is a pervasive challenge in basic cell biology research, drug discovery, and gene delivery. To address this challenge, we have developed a bench-top system comprised of a control interface, that mates to disposable 96-well-formatted microfluidic devices, enabling the individual manipulation, electroporation and real-time monitoring of each cell in suspension. This is the first demonstrated real-time feedback-controlled electroporation of an array of single-cells. Our computer program automatically detects electroporation events and subsequently releases the electric field, precluding continued field-induced damage of the cell, to allow for membrane resealing. Using this novel set-up, we demonstrate the reliable electroporation of an array (n = 15) of individual cells in suspension, using low applied electric fields (<1 V) and the rapid and localized intracellular delivery of otherwise impermeable compounds (Calcein and Orange Green Dextran). Such multiplexed electrical and optical measurements as a function of time are not attainable with typical electroporation setups. This system, which mounts on an inverted microscope, obviates many issues typically associated with prototypical microfluidic chip setups and, more importantly, offers well-controlled and reproducible parallel pressure and electrical application to individual cells for repeatability.
TL;DR: Two commonly used methods to transform Dictyostelium cells are described: calcium phosphate precipitation, resulting in high copy number transformants; and electroporation, an effective technique for producing single integration events into genomic DNA.
Abstract: DNA-mediated transformation is one of the most widely used techniques to study gene function. The eukaryote Dictyostelium discoideum is amenable to numerous genetic manipulations that require insertion of foreign DNA into cells. Here we describe two commonly used methods to transform Dictyostelium cells: calcium phosphate precipitation, resulting in high copy number transformants; and electroporation, an effective technique for producing single integration events into genomic DNA. Single integrations are required for gene disruption by homologous recombination. We also discuss how different selection markers affect vector copy number in transformants and explain why blasticidin has become the preferred selectable marker for making gene knockouts. Both procedures can be accomplished in less than 2 h of hands-on time; however, the calcium phosphate precipitation method contains several incubations, including one of at least 4 h, so the total time required for the transformation is approximately 8 h.
TL;DR: In this paper, the onset or termination of expression of an electroporated DNA can be precisely controlled by timing the administration of tetracycline into an egg, which can reveal previously unknown roles of RhoA, cMeso-1 and Pax2 in early somitogenesis.
TL;DR: It is hypothesized that, not only are aqueous pores created within the lipid membranes, but that nanoscale membrane fragmentation occurs, possibly with micelle formation, which would produce conductivity increases beyond simple electroporation and display a relatively fast turn-off with external voltage.
TL;DR: Findings suggest that electroporation primarily transports molecules across the plasma membrane because its mechanism is specific to lipid bilayer disruption, whereas sonication transfers molecules across both the plasma membranes and cell wall, because it nonspecifically disrupts cell-surface barriers.
Abstract: To assess the cell wall's role as a barrier to intracellular delivery, wild-type Chlamydomonas reinhardtii algal cells and mutant cells lacking a cell wall were exposed to electroporation or sonication. Flow cytometry determined intracellular uptake of calcein and bovine serum albumin (BSA) and loss of cell viability as functions of electroporation transmembrane potential and acoustic energy. Electroporation of wild-type cells increased calcein uptake with increasing transmembrane potential, but delivered much less BSA. Electroporation of wall-deficient cells had similar effects on calcein uptake, but increased BSA uptake as much as 7.5-fold relative to wild-type cells, which indicated that the cell wall was a significant barrier to BSA delivery during electroporation. Sonication of wild-type cells caused calcein and BSA uptake at similar levels. This suggests that the cell wall barrier to BSA delivery can be overcome by sonication. Increased electroporation transmembrane potential or acoustic energy also caused increased loss of cell viability, where wall-deficient cells were especially susceptible to lysis. Overall, we believe this is the first study to compare directly the effects of electroporation and sonication in any cell type. Specifically, these findings suggest that electroporation primarily transports molecules across the plasma membrane because its mechanism is specific to lipid bilayer disruption, whereas sonication transports molecules across both the plasma membrane and cell wall, because it nonspecifically disrupts cell-surface barriers.
TL;DR: A new micro electroporation cell chip with three-dimensional (3D) electrodes was fabricated by means of MEMS technology, and tested on cervical cancer (HeLa) cells, and it was found that, when the molecular size increases, the corresponding Electroporation boundary becomes closer to the electric cell lysis boundary.
TL;DR: Electroporation parameters were optimized for transgene expression with minimal tissue damage with a novel electrode and offers the potential for easier and more reproducible electrically mediated cutaneous plasmid delivery than the simple electrodes currently commercially available.
Abstract: The easy accessibility of skin makes it an excellent target for gene transfer protocols. To take advantage of skin as a target for gene transfer, it is important to establish an efficient and reproducible delivery system. Electroporation is an established technique for enhancing plasmid delivery to many tissues in vivo. A critical component of this technique is the electrode configuration. Electroporation parameters were optimized for transgene expression with minimal tissue damage with a novel electrode. The highest transgene expression and efficiency of individual cell transformation with minimal damage was produced with eight 150 ms pulses at field strength of 100 V/cm. This electrode design offers the potential for easier and more reproducible electrically mediated cutaneous plasmid delivery than the simple electrodes currently commercially available. This electrode can be a valuable tool in determining the applicability of electrically mediated cutaneous gene transfer.
TL;DR: A fast protocol that can be used to obtain highly purified cultures of proliferating adult human and rat Schwann cells accessible for non-viral transfection methods and results in Schwann cell cultures that are more than 90–95% pure.
Abstract: We present a fast protocol that can be used to obtain highly purified cultures of proliferating adult human and rat Schwann cells accessible for non-viral transfection methods. The use of enriched genetically modified adult Schwann cells is of interest in the context of autologous cell transplantation within nerve transplants for peripheral nerve repair. Cell preparation from pre-degenerated adult peripheral nerves is described, together with the use of melanocyte growth medium plus forskolin, fibroblast growth factor-2 (FGF-2), pituitary extract and heregulin as a selective, serum-free culture medium and a subsequent cell enrichment step (cold jet). Proliferating adult Schwann cells can be efficiently genetically modified using optimized, non-viral electroporation protocols. The protocol results in Schwann cell cultures that are more than 90-95% pure, and transfection efficiencies vary depending on the initial cell constitution from 20 to 40%. The procedure takes up to 21 d, depending on the length of the pre-degeneration period.
TL;DR: Logistic regression was used to determine whether the probabilities of cell survival and electroporation depend on experimental conditions and cell properties and found that cells are more readily permeabilized and are more likely to survive if they are large and hemispherical as opposed to small and ellipsoidal with a high aspect ratio.
Abstract: Single-cell electroporation was performed using electrolyte-filled capillaries on fluorescently labeled A549 cells. Cells were exposed to brief pulses (50-300 ms) at various cell-capillary tip distances. Cell viability and electroporation success were measured. In order to understand the variability in single-cell electroporation, logistic regression was used to determine whether the probabilities of cell survival and electroporation depend on experimental conditions and cell properties. Both experimental conditions and cell properties (size and shape) have a significant effect on the outcome. Finite element simulations were used to compare bulk electroporation to single-cell electroporation in terms of cell size and shape. Cells are more readily permeabilized and are more likely to survive if they are large and hemispherical as opposed to small and ellipsoidal with a high aspect ratio. The dependence of the maximum transmembrane potential across the cell membrane on cell size is much weaker than it is for bulk electroporation. Observed survival probabilities are related to the calculated fraction of the cell's surface area that is electroporated. Observed success of electroporation is related to the maximum transmembrane potential achieved.
TL;DR: Intadermal DNA electrotransfer into the skin, the muscle and the ear pinna all resulted in IFN-gamma secretion by luciferase-stimulated splenocytes suggesting that an efficient Th1 response was induced in all case.
TL;DR: Low frequency UDD can be readily scaled up, allowing for the application of UDD not only in laboratory conditions but also on an industrial scale.
Abstract: In environmental microbiology, the most commonly used methods of bacterial DNA transfer are conjugation and electroporation. However, conjugation requires physical contact and cell–pilus–cell interactions; electroporation requires low-ionic strength medium and high voltage. These limitations have hampered broad applications of bacterial DNA delivery. We have employed a standard low frequency 40kHz ultrasound bath to successfully transfer plasmid pBBR1MCS2 into Pseudomonas putida UWC1, Escherichia coli DH5a and Pseudomonas fluorescens SBW25 with high efficiency. Under optimal conditions: ultrasound exposure time of 10s, 50mM CaCl2, temperature of 228C, plasmid concentration of 0.8ng/ml, P. putida UWC1 cell concentration of 2.5 � 10 9 CFU (colony forming unit)/ml and reaction volume of 500ml, the efficiency of ultrasound DNA delivery (UDD) was 9.8 � 2.3 � 10 � 6 transformants per cell, which was nine times more efficient than conjugation, and even four times greater than electroporation. We have also transferred pBBR1MCS2 into E. coli DH5a and P. fluorescens SBW25 with efficiencies of 1.16 � 0.13 � 10 � 6 and 4.33 � 0.78 � 10 � 6 transformants per cell, respectively. Low frequency UDD can be readily scaled up, allowing for the application of UDD not only in laboratory conditions but also on an industrial scale.
TL;DR: Electroporation can be used as a versatile tool to investigate molecular pathways involved in axon extension during Xenopus embryogenesis and can be tailored easily to other developing organ systems and to other organisms by making simple adjustments to the electroporation chamber.
Abstract: Background
Blastomere injection of mRNA or antisense oligonucleotides has proven effective in analyzing early gene function in Xenopus. However, functional analysis of genes involved in neuronal differentiation and axon pathfinding by this method is often hampered by earlier function of these genes during development. Therefore, fine spatio-temporal control of over-expression or knock-down approaches is required to specifically address the role of a given gene in these processes.
TL;DR: It is demonstrated that reporter genes introduced into the developing mouse cerebral cortex by in utero electroporation can achieve promoter/enhancer‐specific patterns of expression and it is shown that the DCX promoter identified via Electroporation could reproduce doublecortin expression in the entire central nervous system in DCX‐DsRed‐express transgenic mice.
Abstract: Characterization of neural promoter/enhancers is essential for understanding gene regulation during brain development and provides useful genetic tools. However, it relies on the use of transgenic mice. We report a method for the rapid in vivo analysis of neural promoter/enhancers in the developing mouse brain and its application in the isolation of the doublecortin (DCX) promoter/enhancer for genetic labeling of young neurons. In the present study, we demonstrated that reporter genes introduced into the developing mouse cerebral cortex by in utero electroporation can achieve promoter/enhancer-specific patterns of expression. We used the in utero electroporation system to isolate a genomic fragment of the doublecortin gene that can direct reporter expression faithful to doublecortin in young neurons of the cerebral cortex. Finally, we showed that the DCX promoter identified via electroporation could reproduce doublecortin expression in the entire central nervous system in DCX-DsRed-express transgenic mice. The results of our study provide a convenient, reliable, and rapid method for in vivo analysis of neural promoter/enhancers in the developing mouse brain.
TL;DR: A multi-channel electroporation microchip made of polydimethylsiloxane (PDMS) and glass for gene transfer in mammalian cells that was able to produce five different transfection rates and survival rates at different electric fields produced in a single microchip is developed.
TL;DR: It is found that use of electroporation allows a single intramuscular DNA injection to be as effective as repeated i.m. DNA injections in activation of both Ag85A-specific CD4 and CD8 T cells and co-expression of immune-enhancing cytokine GM-CSF by the same plasmid DNA TB vaccine could further enhance T cell activation.
TL;DR: A longitudinal in vitro evaluation of cellular viability, apoptosis, proliferation, and cardiac differentiation of magnetically labeled mESC was conducted and no significant difference was seen in these biological parameters among the three transfection methods.
Abstract: In vivo MRI of stem cells (SCs) is an emerging application to evaluate the role of cell therapy in restoring the injured myocardium. The high spatial and temporal resolution combined with iron-oxide-based intracellular labeling techniques will provide a sensitive, noninvasive, dual imaging modality for both cells and myocardium. In order to facilitate this novel imaging approach, much effort has been directed towards developing efficient transfection methods. While techniques utilizing poly-L-lysine (PLL), protamine sulfate (PS), and electroporation (ELP) have been proposed, the fundamental biological effects of these methods on mouse embryonic SCs (mESC) have not been investigated systematically. In this study a longitudinal in vitro evaluation of cellular viability, apoptosis, proliferation, and cardiac differentiation of magnetically labeled mESC was conducted. No significant difference was seen in these biological parameters among the three transfection methods. However, cardiac differentiation was most attenuated by ELP, and iron uptake was most effective by PS.
TL;DR: It is demonstrated that electroporation can be used effectively in healthy and injured lungs to facilitate gene delivery and expression and is the first successful use of gene delivery to treat existing lung injury.
Abstract: Rationale: Acute lung injury and acute respiratory distress syndrome are common clinical syndromes resulting largely from the accumulation of and inability to clear pulmonary edema, due to injury to the alveolar epithelium. Gene therapy may represent an important alternative for the treatment and prevention of these diseases by restoring alveolar epithelial function. We have recently developed an electroporation strategy to transfer genes to the lungs of mice, with high efficiency and low inflammation.
Objectives: We asked whether electroporation-mediated transfer of genes encoding subunits of the Na+,K+-ATPase could protect from LPS-induced lung injury or be used to treat already injured lungs by up-regulating mechanisms of pulmonary edema clearance.
Methods: Plasmids were delivered to the lungs of mice using transthoracic electroporation. Lung injury was induced by intratracheal administration of LPS (4 mg/kg body weight). Biochemical, cellular, and physiologic measurements were taken to assess gene transfer and lung injury.
Measurements and Main Results: Improvements in wet-to-dry ratios, pulmonary effusions, bronchoalveolar lavage protein levels and cellularity, alveolar fluid clearance, and respiratory mechanics were seen after delivery of plasmids expressing Na+,K+-ATPase subunits, but not control plasmids, in LPS-injured lungs. Delivery of plasmids expressing Na+,K+-ATPase subunits both protected from subsequent lung injury and partially reversed existing lung injury by these measures.
Conclusions: These results demonstrate that electroporation can be used effectively in healthy and injured lungs to facilitate gene delivery and expression. To our knowledge, this is the first successful use of gene delivery to treat existing lung injury, and may have future clinical potential.