RESUMEN
Pancreatic cancer (PC) is a lethal disease with extremely high mortality. Although surgical resection is the optimal therapeutic approach for PC, about 30%-40% of those patients are not candidates for surgical resection when diagnosed. Chemotherapy and radiotherapy also could not claim a desirable effect on PC. The application of interventional radiology approaches is limited by unavoidable damage to the surrounding vessels or organs. By the superiority of mechanism and technology, IRE could ablate the tumor by creating irreversible pores on the membrane of PC cells with other tissues like vessels and pancreatic ducts untouched. This consensus gathers the theoretical basis and clinical experience from multiple Chinese medical centers, to provide the application principles and experience from Chinese experts in the IRE field.
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Técnicas de Ablación/normas , Electroporación/normas , Neoplasias Pancreáticas/cirugía , Guías de Práctica Clínica como Asunto , Cirugía Asistida por Computador/normas , Técnicas de Ablación/métodos , China , Consenso , Electroporación/métodos , Testimonio de Experto , Humanos , Páncreas/diagnóstico por imagen , Páncreas/cirugía , Neoplasias Pancreáticas/diagnóstico , Cirugía Asistida por Computador/métodos , Resultado del TratamientoRESUMEN
CAR-T therapy is a particularly effective treatment for some types of cancer that uses retroviruses to deliver the gene for a chimeric antigen receptor (CAR) to a patient's T cells ex vivo. The CAR enables the T cells to bind and eradicate cells with a specific surface marker (e.g., CD19+ B cells) after they are transfused back into the patient. This treatment was proven to be particularly effective in treating non-Hodgkin's lymphoma (NHL) and acute lymphoblastic leukemia (ALL), but the current CAR-T cell manufacturing process has a few significant drawbacks. For example, while lentiviral and gammaretroviral transduction are both relatively effective, the process of producing viral vectors is time-consuming and costly. Additionally, patients must undergo follow up appointments for several years to monitor them for any unanticipated side effects associated with the virus. Therefore, several studies have endeavored to find alternative non-viral gene delivery methods that are less expensive, more precise, simple, and safe. This review focuses on the current state of the most promising non-viral gene delivery techniques, including electroporation and transfection with cationic polymers or lipids.
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Electroporación/normas , Terapia Genética/métodos , Inmunoterapia Adoptiva/métodos , Linfoma no Hodgkin/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Linfocitos T/inmunología , Animales , Electroporación/métodos , Humanos , Linfoma no Hodgkin/inmunología , Linfoma no Hodgkin/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologíaRESUMEN
OBJECTIVE: The objective was to evaluate the feasibility and safety of computed tomography (CT)-guided percutaneous irreversible electroporation (IRE) in porcine kidneys. MATERIALS AND METHODS: Under CT guidance, two monopole probes were used to precisely puncture through the renal parenchyma into the renal hilum in nine anesthetized adult Bama miniature pigs. After which, IRE ablation was performed. Biochemical and pathological examinations were carried out 2 h, 2, 7, and 14 days after the procedure. RESULTS: All procedures were performed successfully without any serious complications such as bleeding, infection, or death. All pigs survived until the end of the study. Pathological examinations showed that cells in the ablation area were dead within 2 days after the procedure, whereas the vascular endothelium showed only slight damage. After 2 days, endothelialization ensued and regrowth of smooth muscle cells was observed after 14 days. Hemogram tests indicated a transient increase but gradually returned to baseline levels 14 days after the procedure. CONCLUSION: IRE was essentially safe, however further studies on tumor ablation using several different animal models are needed.
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Electroporación/normas , Riñón/cirugía , Cirugía Asistida por Computador/métodos , Tomografía Computarizada por Rayos X/métodos , Técnicas de Ablación/métodos , Animales , Forma MB de la Creatina-Quinasa/sangre , Electroporación/métodos , Estudios de Factibilidad , Hidroxibutirato Deshidrogenasa/sangre , Riñón/metabolismo , Riñón/patología , L-Lactato Deshidrogenasa/sangre , Leucocitos/patología , Modelos Animales , Porcinos , Resultado del TratamientoRESUMEN
PURPOSE: A consensus study of panelists was performed to provide a uniform protocol regarding (contra) indications, procedural parameters, perioperative care, and follow-up of irreversible electroporation (IRE) for the treatment of hepatic malignancies. MATERIALS AND METHODS: Interventional radiologists who had 2 or more publications on IRE, reporting at least 1 patient cohort in the field of hepatobiliary IRE, were recruited. The 8 panelists were asked to anonymously complete 3 iterative rounds of IRE-focused questionnaires to collect data according to a modified Delphi technique. Consensus was defined as having reached 80% or greater agreement. RESULTS: Panel members' response rates were 88%, 75%, and 88% in rounds 1, 2, and 3, respectively; consensus was reached on 124 of 136 items (91%). Percutaneous or intraoperative hepatic IRE should be considered for unresectable primary and secondary malignancies that are truly unsuitable for thermal ablation because of proximity to critical structures. Absolute contraindications are ventricular arrhythmias, cardiac stimulation devices, and congestive heart failure of New York Heart Association class 3 or higher. A metal stent outside the ablation zone should not be considered a contraindication. For the only commercially available IRE device, the recommended settings are an inter-electrode distance of 10-20 mm and an exposure length of 20 mm. After 10 test pulses, 90 treatment pulses of 1500 V/cm should be delivered continuously, with a pulse length of 70-90 µs. The first post-procedural follow-up should take place 1 month after IRE and thereafter every 3 months, using cross-sectional imaging plus tumor marker assessment. CONCLUSIONS: This article provides recommendations, created by a modified Delphi consensus study, regarding patient selection, workup, procedure, and follow-up of IRE treatment for hepatic malignancies.
Asunto(s)
Técnicas de Ablación/normas , Electroporación/normas , Neoplasias Hepáticas/cirugía , Técnicas de Ablación/efectos adversos , Toma de Decisiones Clínicas , Consenso , Contraindicaciones de los Procedimientos , Técnica Delphi , Medicina Basada en la Evidencia , Humanos , Neoplasias Hepáticas/patología , Selección de Paciente , Factores de RiesgoRESUMEN
Irreversible electroporation has raised great interest in the past decade as a means of destroying cancers in a way that does not involve heat. Irreversible electroporation is a novel ablation technology that uses short high-voltage electrical pulses to enhance the permeability of tumor cell membranes and generate irreversible nano-sized structural defects or pores, thus leading to cell death. Irreversible electroporation has many advantages over thermal therapies due to its nonthermal mechanism: (1) reduced risk of injury to surrounding organs and (2) no "heat-sink" effect due to nearby blood vessels. However, so far, it has been difficult for irreversible electroporation to completely ablate large tumors (eg, >3 cm in diameter). In order to overcome this problem, many preclinical and clinical studies have been performed to improve the efficacy of IRE in the treatment of large size of tumors through a chemical perspective. Due to the distribution of electric field, irreversible electroporation region, reversible electroporation region, and intact region can be found in the treatment of irreversible electroporation. Thus, 2 types of chemical enhancements of irreversible electroporation were discussed in the article, such as the reversible electroporation region enhanced and the irreversible electroporation region enhanced. Specifically, the state-of-the-art results regarding the following approaches that have the potential to be used in the enhancement of irreversible electroporation were systematically reviewed in the article, including (1) combination with cytotoxic drugs, (2) calcium electroporation, (3) modification of cell membrane, and (4) modification of the tumor cell microenvironment. In the end, we concluded with 4 issues that should be addressed in the future for improving irreversible electroporation further in a chemical way.
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Electroporación/métodos , Algoritmos , Animales , Antineoplásicos/farmacología , Calcio/farmacología , Supervivencia Celular/efectos de los fármacos , Microambiente Celular/efectos de los fármacos , Electroporación/normas , Humanos , Modelos Teóricos , Tensoactivos/farmacologíaRESUMEN
The gene delivery to skeletal muscles is a promising strategy for the treatment of both muscular disorders (by silencing or overexpression of specific gene) and systemic secretion of therapeutic proteins. The use of a physical method like electroporation with plate or needle electrodes facilitates long-lasting gene silencing in situ. It has been reported that electroporation enhances the expression of the naked DNA gene in the skeletal muscle up to 100 times and decreases the changeability of the intramuscular expression. Coelectransfer of reporter genes such as green fluorescent protein (GFP), luciferase or beta-galactosidase allows the observation of correctly performed silencing in the muscles. Appropriate selection of plasmid injection volume and concentration, as well as electrotransfer parameters, such as the voltage, the length and the number of electrical pulses do not cause long-term damage to myocytes. In this review, we summarized the electroporation methodology as well as the procedure of electrotransfer to the gastrocnemius, tibialis, soleus and foot muscles and compare their advantages and disadvantages.
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Electroporación/métodos , Técnicas de Transferencia de Gen/normas , Músculo Esquelético/metabolismo , Animales , Electroporación/normas , Técnicas de Transferencia de Gen/efectos adversos , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Plásmidos/genética , Plásmidos/metabolismoRESUMEN
In this study we evaluated the influence of medium conductivity to propidium iodide (PI) and bleomycin (BLM) electroporation mediated transfer to cells. Inverse dependency between the extracellular conductivity and the efficiency of the transfer had been found. Using 1 high voltage (HV) pulse, the total molecule transfer efficiency decreased 4.67 times when external medium conductivity increased from 0.1 to 0.9 S/m. Similar results had been found using 2 HV and 3 HV pulses. The percentage of cells killed by BLM electroporation mediated transfer had also decreased with the conductivity increase, from 79% killed cells in 0.1 S/m conductivity medium to 28% killed cells in 0.9 S/m conductivity medium. We hypothesize that the effect of external medium conductivity on electroporation mediated transfer is triggered by cell deformation during electric field application. In high conductivity external medium cell assumes oblate shape, which causes a change of voltage distribution on the cell membrane, leading to lower electric field induced transmembrane potential. On the contrary, low conductivity external medium leads to prolate cell shape and increased transmembrane potential at the electrode facing cell poles.
Asunto(s)
Medios de Cultivo/química , Electroporación/métodos , Animales , Bleomicina/química , Células CHO , Supervivencia Celular , Cricetinae , Cricetulus , Conductividad Eléctrica , Electroporación/normas , Propidio/químicaRESUMEN
La electroquimioterapia (EQT) es una modalidad de tratamiento de lesiones cutáneas y subcutáneas originadas por melanoma u otros tumores. El procedimiento consiste en la administración de agentes antineoplásicos, seguido de impulsos eléctricos locales, para conseguir un efecto conocido como electroporación, que permite la entrada al citosol de medicamentos que no difunden a través de la membrana celular. El objetivo de esta revisión es establecer la evidencia que sustenta la incorporación de la EQT como estrategia terapéutica en el melanoma. Además, se ha llevado a cabo una revisión sistemática de la literatura con síntesis cualitativa. Se ha realizado una búsqueda cualificada de la literatura en bases de datos referenciales y a texto completo. Fueron seleccionados 7 estudios: 3 revisiones sistemáticas y 4 series de casos. La calidad de la evidencia encontrada no es buena, pero la coincidencia de sus resultados en algunas las variables le da consistencia. Los metaanálisis muestran resultados a favor de la EQT frente a la quimioterapia. La EQT parece un procedimiento efectivo en el tratamiento local de nódulos tumorales malignos (nivel medio o bajo de calidad de la evidencia). Es un tratamiento fácil de administrar, económico y bien tolerado con el que se consigue respuesta objetiva en circunstancias determinadas. No hay evidencia de que pueda afectar el curso natural de la enfermedad, por lo que debe considerarse un tratamiento paliativo. Con un nivel de la evidencia 1- (1 menos), puede recomendarse la incorporación de la EQT para el tratamiento paliativo del melanoma locorregionalmente avanzado irresecable (fuerza de la recomendación: B)
Electrochemotherapy is a therapeutic option for the treatment of cutaneous and subcutaneous metastases from melanoma and other tumors. The procedure consists of the administration of anticancer drugs followed by locally applied electrical impulses to achieve an effect known as electroporation, which facilitates entry into the cytosol of drugs that cannot cross the cell membrane. The aim of this review is to evaluate the evidence that supports the use of electrochemotherapy as a therapeutic strategy in melanoma. We conducted a qualitative systematic review of the literature using advanced searches of bibliographic databases and full text reviews. Seven studies (3 systematic reviews and 4 cases series) were selected. The quality of the evidence was not good, but the coincidence of results for certain variables supports their consistency. Results of the meta-analyses favored electrochemotherapy over chemotherapy. Electrochemotherapy appears to be an effective procedure for the local treatment of malignant tumor nodules (evidence of intermediate or low quality). This inexpensive method is simple to apply, well tolerated, and achieves objective responses under certain circumstances. There is no evidence that electrochemotherapy alters the natural course of the disease and it should therefore be considered a palliative treatment. With an evidence level of 1- (minus), electrochemotherapy can be recommended for the palliative treatment of unresectable, locoregionally advanced melanoma (grade B recommendation)
Asunto(s)
Humanos , Masculino , Femenino , Melanoma/tratamiento farmacológico , Electroquimioterapia/instrumentación , Electroquimioterapia/métodos , Electroporación/métodos , Electroporación , Electroquimioterapia/normas , Electroquimioterapia , Electroporación/normas , Electroporación/tendencias , Cuidados Paliativos/tendenciasRESUMEN
Irreversible Electroporation (IRE) is used to treat locally advanced cancers, commonly of the pancreas, liver, kidney, and other soft tissues. Precise eligibility for IRE should be established in each individual patient by a multidisciplinary team based on comprehensive clinical, imaging, and laboratory assessment. Standardization of IRE technique and protocols is expected to improve safety, lead to reproducible outcomes, and facilitate further research into IRE. The present article provides a set of technical recommendations for the use of IRE in the treatment of locally advanced pancreatic cancer. J. Surg. Oncol. 2016;114:865-871. © 2016 2016 Wiley Periodicals, Inc.
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Adenocarcinoma/terapia , Electroporación/métodos , Neoplasias Pancreáticas/terapia , Adenocarcinoma/patología , Cuidados Posteriores , Electroporación/normas , Humanos , Neoplasias Pancreáticas/patología , Selección de PacienteRESUMEN
Cell penetrating peptides (CPPs) have been used for a myriad of cellular delivery applications and were recently explored for delivery of antisense agents such as peptide nucleic acids (PNAs) for bacterial inhibition. Although these molecular systems (i.e. CPP-PNAs) have shown ability to inhibit growth of bacterial cultures in vitro, they show limited effectiveness in killing encapsulated intracellular bacteria in mammalian cells such as macrophages, presumably due to difficulty involved in the endosomal escape of the reagents. In this report, we show that electroporation delivery dramatically increases the bioavailability of CPP-PNAs to kill Salmonella enterica serovar Typhimurium LT2 inside macrophages. Electroporation delivers the molecules without involving endocytosis and greatly increases the antisense effect. The decrease in the average number of Salmonella per macrophage under a 1200 V cm(-1) and 5 ms pulse was a factor of 9 higher than that without electroporation (in an experiment with a multiplicity of infection of 2 : 1). Our results suggest that electroporation is an effective approach for a wide range of applications involving CPP-based delivery. The microfluidic format will allow convenient functional screening and testing of PNA-based reagents for antisense applications.
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Sistemas de Liberación de Medicamentos/métodos , Electroporación/normas , Oligopéptidos/farmacología , Ácidos Nucleicos de Péptidos/farmacología , Salmonella typhimurium/crecimiento & desarrollo , Animales , Línea Celular , Recuento de Colonia Microbiana , Sistemas de Liberación de Medicamentos/normas , Electroporación/instrumentación , Ratones , MicrofluídicaRESUMEN
BACKGROUND: Many variations in avian in ovo transfection of the neural tube/crest have been reported, but never compared quantitatively. RESULTS: Genome integrating pT2K-CAGGS-GFP and pCAGGS-T2TP transposase plasmids were co-electroporated into quail E2 embryo trunk neural tube and the proportion of GFP-expressing neural cells was counted 1 and 7 days later. Electroporation efficiency increased with plasmid concentration and pulse number but plateaued at, respectively, above 1.25 µg/µL and 3 pulses. Bilateral electroporation transfected more cells than unilateral but less than that anticipated by doubling the unilateral treatment. Holding the concentration of GFP plasmid constant and varying the transposase plasmid concentration revealed an optimum ratio of, in this case, 4:1 (1.2 µg/µL:0.3 µg/µL). Leaving transfected embryos to E9 confirmed that expression was maintained in vivo with the transposase system, but declined with non-integrated plasmid. Transfection of neural crest cells was low if electroporated less than 6-8 hr before emigration. We propose this indicates loss of epithelial integrity well prior to exit. We suggest this event be termed epithelio-mesenchymal transition sensu stricto, whereas the term delamination be reserved for the later emigration from the neural epithelium. CONCLUSIONS: Co-electroporation in ovo must take into account plasmid(s) concentration and ratio, pulse number, pulse directionality, and timing.
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Electroporación/métodos , Cresta Neural/metabolismo , Tubo Neural/metabolismo , Codorniz/embriología , Transfección/métodos , Cigoto/metabolismo , Análisis de Varianza , Animales , Recuento de Células , Células Cultivadas , Electroporación/normas , Proteínas Fluorescentes Verdes/metabolismo , Tubo Neural/citología , Transposasas/metabolismoRESUMEN
Chick in ovo neural tube electroporation has become a widely used method for assaying gene function during embryonic development. Since its first description, many variants of this technique have been described, with varying values for specific parameters such as electrode type and spacing, voltage, pulse duration and plasmid DNA concentration. Here we examine the influence of some of these variables and derive a detailed and optimal protocol for electroporating the caudal neural tube during the third day of embryonic development. Our findings highlight the importance of electrode placement and DNA dilution buffer for optimal expression and absence of electroporation artifacts.
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Embrión de Pollo/cirugía , Electroporación/métodos , Electroporación/normas , Técnicas de Transferencia de Gen/normas , Tubo Neural/cirugía , Animales , Cadherinas/genética , Cadherinas/metabolismo , Embrión de Pollo/embriología , Electroporación/instrumentación , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Técnicas de Transferencia de Gen/instrumentación , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Microelectrodos/normas , Tubo Neural/embriología , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Médula Espinal/citología , Médula Espinal/embriología , Médula Espinal/metabolismoRESUMEN
Electroporation, a non-virus-mediated gene transfection method, has traditionally had poor outcomes with low gene transfection efficiency and poor cellular viability, particularly in primary human lymphocytes. Herein we have optimized the electroporation conditions for primary CD8+ cells resulting in a maximum rate of 81.3%, and a mean transfection efficiency of 59.6%. After removal of dead cells, the viability of transfected primary CD8+ cells was greater than 90%, similar to untransfected controls. Using this procedure, primary human CD8+ cells transfected with an interferon α8 plasmid produced fluids that inhibited HIV-1 replication by > 95%. This transfection protocol is useful for transfection of other primary blood cells, such as CD4+ T cells, and for studying the function of genes in primary human blood cells instead of cell lines. The transfection procedure also has potential application in gene therapy clinical trials to treat diseases utilizing transfected primary human cells.
Asunto(s)
Linfocitos T CD8-positivos/fisiología , Electroporación/métodos , Interferón-alfa/genética , Transfección/métodos , Linfocitos T CD8-positivos/inmunología , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Electroporación/normas , Citometría de Flujo , Terapia Genética/métodos , Terapia Genética/normas , VIH-1/genética , VIH-1/inmunología , Humanos , Interferón-alfa/inmunología , Microscopía Confocal , Plásmidos/genética , Plásmidos/inmunología , Transfección/normasRESUMEN
BACKGROUND: DNA-based vaccines have been safe but weakly immunogenic in humans to date. METHODS AND FINDINGS: We sought to determine the safety, tolerability, and immunogenicity of ADVAX, a multigenic HIV-1 DNA vaccine candidate, injected intramuscularly by in vivo electroporation (EP) in a Phase-1, double-blind, randomized placebo-controlled trial in healthy volunteers. Eight volunteers each received 0.2 mg, 1 mg, or 4 mg ADVAX or saline placebo via EP, or 4 mg ADVAX via standard intramuscular injection at weeks 0 and 8. A third vaccination was administered to eleven volunteers at week 36. EP was safe, well-tolerated and considered acceptable for a prophylactic vaccine. EP delivery of ADVAX increased the magnitude of HIV-1-specific cell mediated immunity by up to 70-fold over IM injection, as measured by gamma interferon ELISpot. The number of antigens to which the response was detected improved with EP and increasing dosage. Intracellular cytokine staining analysis of ELISpot responders revealed both CD4+ and CD8+ T cell responses, with co-secretion of multiple cytokines. CONCLUSIONS: This is the first demonstration in healthy volunteers that EP is safe, tolerable, and effective in improving the magnitude, breadth and durability of cellular immune responses to a DNA vaccine candidate. TRIAL REGISTRATION: ClinicalTrials.gov NCT00545987.
Asunto(s)
Vacunas contra el SIDA/administración & dosificación , Electroporación/métodos , VIH-1/inmunología , Inmunidad Celular/efectos de los fármacos , Vacunas de ADN/administración & dosificación , Vacunas contra el SIDA/farmacología , Adolescente , Adulto , Citocinas/metabolismo , Método Doble Ciego , Electroporación/normas , Femenino , Humanos , Inyecciones Intramusculares , Masculino , Persona de Mediana Edad , Linfocitos T/inmunología , Vacunas de ADN/farmacología , Adulto JovenRESUMEN
BACKGROUND: The antigen-specific immune response is dependent not only on the properties of the antigens, but also on their encounter with antigen-presenting cells. A previous study showed that the spleen produced a large amount of transgenes after direct tissue injection of plasmid DNA. In addition, the spleen is the largest organ in the lymphatic system and contains a variety of types of immune cells, including lymphocytes, macrophages and dendritic cells. Thus, it can be a promising target for DNA vaccination. METHODS: Tissue-dependent properties of transgene expression were examined using a plasmid vector expressing firefly luciferase. Mice received injections of pCMV-Luc into the dorsal skin or spleen followed by electroporation, and the luciferase activity was measured 6 h after injection. Then, plasmids expressing a model antigen ovalbumin (pCMV-OVA) or its typical major histocompatibility complex class I-restricted epitope SIINFEKL (pPep-ER) were injected into C57BL/6 mice twice at an interval of 1 week. Seven days after the second immunization, OVA-specific humoral and cellular immune responses were evaluated. RESULTS: The spleen produced a larger amount of transgenes than the skin after direct tissue injection of plasmid DNA. However, intradermal injection of plasmid DNA resulted in a larger amount of OVA-specific antibodies and a greater cytotoxic T lymphocyte response compared to intrasplenic injection. In addition, intradermal immunization with either pCMV-OVA or pPep-ER generated more protective effects against EG7-OVA tumor challenge. CONCLUSIONS: The results obtained in the present study indicate that the spleen is unlikely to be a good target for immunization despite the presence of a large number of lymphocytes and efficient production of transgenes.
Asunto(s)
Piel/inmunología , Bazo/inmunología , Vacunación/métodos , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología , Animales , Formación de Anticuerpos , Electroporación/métodos , Electroporación/normas , Femenino , Inmunoglobulina G/sangre , Inyecciones , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Plásmidos/administración & dosificación , Plásmidos/inmunologíaRESUMEN
Development of a transgenic porcine biomedical research model requires effective delivery of DNA into the donor cell followed by selection of genetically modified somatic cell lines to be used for nuclear transfer. The objective of the current study was 2-fold: (1) to compare the effectiveness of a single 1 ms pulse of different voltages (V; 100, 150, 200, 250, 300, 350) and multiple 1 ms pulses (1, 2, 3, 4 or 5) at 300 V for delivery and expression of super-coiled GFP vector in surviving cells of three fetal fibroblast cell lines, and (2) to determine the ability of these electroporation parameters to produce stably transfected fibroblast colonies following G418 selection. Cell line (P < 0.001) and voltage (P < 0.001) affected DNA delivery into the cell as assessed by GFP expression while survival at 24 h was affected by voltage (P < 0.001) and not by cell line (P = 0.797). Using a single pulse while increasing voltage resulted in the percentage of GFP expressing cells increasing from 3.2 +/- 0.8% to 43.0 +/- 3.4% while survival decreased from 90.5 +/- 8.0% to 44.8 +/- 2.0%. The number of pulses at 300 V significantly affected survival (P < 0.001) and GFP expression (P < 0.001). Survival steadily decreased following 1-5 pulses from 63.2 +/- 6.3% to 3.0 +/- 0.3% with GFP expression of surviving cells increasing from 35.6 +/- 2.67% to 71.4 +/- 6.1%. Electroporation of a selectable marker at a 1:1 copy number ratio to a co-electroporated transgene resulted in 83% of G418 resistant colonies also being PCR positive for the secondary transgene. These electroporation conditions, specifically, three 1 ms pulses of 300 V to 200 muL of 1 x 10(6) cells/mL in the presence of 12.5 mug DNA/mL effectively introduced DNA into somatic cells. The utilization of these conditions produced numerous transgenic fibroblast colonies following G418 selection that when used for somatic cell nuclear transfer resulted in the production of live offspring.
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Electroporación/métodos , Feto/metabolismo , Fibroblastos/metabolismo , Porcinos , Animales , Animales Modificados Genéticamente , Calibración , Células Cultivadas , Clonación de Organismos/métodos , Clonación de Organismos/veterinaria , Resistencia a Medicamentos/genética , Electroporación/normas , Embrión de Mamíferos , Feto/citología , Feto/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Neomicina/farmacología , Técnicas de Transferencia Nuclear/normas , Técnicas de Transferencia Nuclear/veterinaria , Porcinos/embriología , Transfección/métodos , Transfección/normas , TransgenesRESUMEN
This paper describes the fabrication of microarrays that enable the parallel electroporation of small interfering RNAs (siRNAs) into mammalian cells. To optimize the conditions of microarray preparation and electric pulsing, a self-assembled monolayer was formed on a gold electrode, and a cationic polymer was adsorbed by the entire surface of the monolayer. siRNA was then adsorbed by the cationically modified electrode through electrostatic interactions. Human embryonic kidney cells stably transformed with the expression construct of green fluorescent protein (GFP) were used to examine the electric pulse-triggered transfer of GFP-specific siRNA. A single electric pulse was applied to the cells cultured on the electrode at a field strength of 240 V cm(-1). The expression of GFP was significantly suppressed in a sequence-specific manner two days after pulsing. Microscopic observation and flow-cytometric analysis revealed that the expression of GFP was attenuated in the majority of cells in a loading-dependent manner. Moreover, the effect of siRNA could be temporally controlled by changing the culture periods before pulsing. When a micropatterned self-assembled monolayer was used as a platform for loading siRNA in an array format, gene silencing was spatially restricted to the regions where specific siRNA was loaded. From these results, we conclude that array-based electroporation provides an excellent means of individual transfer of siRNAs into mammalian cells for high-throughput gene function studies.
Asunto(s)
Electroporación/métodos , ARN Interferente Pequeño/farmacocinética , Línea Celular , Electrodos , Electroporación/normas , Citometría de Flujo , Silenciador del Gen , Proteínas Fluorescentes Verdes/genética , Humanos , ARN Interferente Pequeño/administración & dosificaciónRESUMEN
Chronic lymphocytic leukemia (CLL) consists of at least two major prognostic subgroups, characterized by different cellular and molecular markers. This observation sparked studies on the function and clinical importance of these markers. In order to address their function adequately, an efficient and reliable method for gene transfer is needed. In this study, we compared efficiency and utility of different gene transfer techniques in CLL. Lenti-, retro- and adenoviral transduction did not yield appreciable numbers of marker gene enhanced green fluorescent protein (EGFP) positive CLL cells, despite various prestimulation protocols. Efficient transgene expression was observed after nucleofection of CLL cells with plasmid DNA, at the expense of low survival rates. After optimization, electroporation of in vitro transcribed mRNA yielded up to 90% EGFP+CLL cells without affecting survival. Transgene expression remained detectable for at least 2 weeks after electroporation. Furthermore, we could demonstrate overexpression of ZAP70 and of a ZAP70-EGFP fusion protein after electroporation with ZAP70 or ZAP70-EGFP mRNA. We conclude that mRNA electroporation is a novel and straightforward method for highly efficient gene transfer in CLL. The application of this technique should facilitate functional studies on CLL cells, as well as clinical research.
Asunto(s)
Electroporación/métodos , Técnicas de Transferencia de Gen , Leucemia Linfocítica Crónica de Células B/patología , ARN Mensajero/administración & dosificación , Biomarcadores , Supervivencia Celular , Células Cultivadas , Electroporación/normas , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Humanos , Proteínas Recombinantes de Fusión/genética , Proteína Tirosina Quinasa ZAP-70/genéticaRESUMEN
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.