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Berneman, Zwi N., Evelien Smits, Peter Ponsaerts, Marc Lenjou, Griet Nijs, Dirk R. Van Bockstaele y Viggo F. Van Tendeloo. "RNA Electroporation as a New Gene Transfer Method in Hematopoietic Progenitor Cells, Mesenchymal Cells and Activated T-Cells." Blood 104, n.º 11 (16 de noviembre de 2004): 5269. http://dx.doi.org/10.1182/blood.v104.11.5269.5269.
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Abstract Electroporation of messenger RNA has become an established method for gene transfer into dendritic cells for immunotherapeutic purposes. When electroporating dendritic cells, this method combines high transient gene expression with low cell mortality without the risk for insertional mutagenesis. Here, we have investigated the potential of mRNA electroporation to induce short-term transgene expression in other cell types such as bone marrow CD34+ progenitor cells, in vitro cultured bone marrow mesenchymal cells and phytohemagglutinin A (PHA)-stimulated peripheral blood T-cells. Transgene expression after electroporation with mRNA encoding the enhanced green fluorescent protein (EGFP) was evaluated by flow cytometry. Flow cytometric analysis 24h after EGFP mRNA electroporation revealed that 35% of fresh uncultured bone marrow-derived CD34+ hematopoietic progenitor cells was efficiently transfected. In the population of in vitro cultured mesenchymal cells, 90% of the viable CD45-CD13+ cells showed detectable EGFP expression. The level of transient EGFP expression in the PHA-stimulated T-cell population was 47% at 24h after electroporation. Importantly, no significant difference of cell mortality between electroporated and non-electroporated cells was observed, due to the mild electroporation procedure ensuring high cell viability. When looking at the kinetics of EGFP expression, we saw that EGFP was still expressed at day 7 after electroporation with highest expression at day 4. Short-term gene introduction by mRNA electroporation in hematopoietic progenitor cells, mesenchymal cells and T-cells could be used to direct the differentiation and/or to modulate the function of these cells. We conclude that mRNA electroporation is an efficient method for short-term gene transfer in CD34+ cells, mesenchymal cells and activated T-cells with potential applications in gene therapy procedures.
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Whyte, Lyle G. y William E. Inniss. "Electroporation and its effect on the psychrotrophic bacterium Bacillus psychrophilus". Canadian Journal of Microbiology 40, n.º 2 (1 de febrero de 1994): 83–89. http://dx.doi.org/10.1139/m94-014.
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The psychrotrophic bacterium Bacillus psychrophilus was successfully transformed by electroporation with the cloning vector pC194 and the expression vector pPL708. Optimal electroporation parameters such as field strength, pulse length, and electroporation medium, as well as the influence of the growth phase of the bacterial cells, were determined. Maximum transformation efficiencies of 104 transformants/μg plasmid DNA were achieved with late logarithmic – early stationary phase cells grown in medium supplemented with 33.3 mM glycine and electroporated at high field strengths of 18–20 kV∙cm−1. By means of this procedure, a kanamycin-resistant gene was directly cloned into pPL708 and the recombinant plasmid electrotransformed into B. psychrophilus. In addition, the effect of electroporation on protein synthesis was analyzed by 2-dimensional polyacrylamide gel electrophoresis and computing scanning laser densitometry. At least eight proteins were induced by electroporation field strengths of 18 or 25 kV during the first 2 h immediately following electroporation. Conversely, the synthesis of at least five proteins was repressed by electroporation.Key words: electroporation, psychrotrophic bacterium.
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Sales Conniff, Amanda, Jared Tur, Kristopher Kohena, Min Zhang, Justin Gibbons y Loree C. Heller. "Transcriptomic Analysis of the Acute Skeletal Muscle Effects after Intramuscular DNA Electroporation Reveals Inflammatory Signaling". Vaccines 10, n.º 12 (29 de noviembre de 2022): 2037. http://dx.doi.org/10.3390/vaccines10122037.
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Skeletal muscle is a promising tissue for therapeutic gene delivery because it is highly vascularized, accessible, and capable of synthesizing protein for therapies or vaccines. The application of electric pulses (electroporation) enhances plasmid DNA delivery and expression by increasing membrane permeability. Four hours after plasmid electroporation, we evaluated acute gene and protein expression changes in mouse skeletal muscle to identify regulated genes and genetic pathways. RNA sequencing followed by functional annotation was used to evaluate differentially expressed mRNAs. Our data highlighted immune signaling pathways that may influence the effectiveness of DNA electroporation. Cytokine and chemokine protein levels in muscle lysates revealed the upregulation of a subset of inflammatory proteins and confirmed the RNA sequencing analysis. Several regulated DNA-specific pattern recognition receptor mRNAs were also detected. Identifying unique molecular changes in the muscle will facilitate a better understanding of the underlying molecular mechanisms and the development of safety biomarkers and novel strategies to improve skeletal muscle targeted gene therapy.
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Chau, Chalmers, Paolo Actis y Eric Hewitt. "Methods for protein delivery into cells: from current approaches to future perspectives". Biochemical Society Transactions 48, n.º 2 (8 de abril de 2020): 357–65. http://dx.doi.org/10.1042/bst20190039.
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The manipulation of cultured mammalian cells by the delivery of exogenous macromolecules is one of the cornerstones of experimental cell biology. Although the transfection of cells with DNA expressions constructs that encode proteins is routine and simple to perform, the direct delivery of proteins into cells has many advantages. For example, proteins can be chemically modified, assembled into defined complexes and subject to biophysical analyses prior to their delivery into cells. Here, we review new approaches to the injection and electroporation of proteins into cultured cells. In particular, we focus on how recent developments in nanoscale injection probes and localized electroporation devices enable proteins to be delivered whilst minimizing cellular damage. Moreover, we discuss how nanopore sensing may ultimately enable the quantification of protein delivery at single-molecule resolution.
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Campillo-Davo, Diana, Maxime De Laere, Gils Roex, Maarten Versteven, Donovan Flumens, Zwi N. Berneman, Viggo F. I. Van Tendeloo, Sébastien Anguille y Eva Lion. "The Ins and Outs of Messenger RNA Electroporation for Physical Gene Delivery in Immune Cell-Based Therapy". Pharmaceutics 13, n.º 3 (16 de marzo de 2021): 396. http://dx.doi.org/10.3390/pharmaceutics13030396.
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Messenger RNA (mRNA) electroporation is a powerful tool for transient genetic modification of cells. This non-viral method of genetic engineering has been widely used in immunotherapy. Electroporation allows fine-tuning of transfection protocols for each cell type as well as introduction of multiple protein-coding mRNAs at once. As a pioneering group in mRNA electroporation, in this review, we provide an expert overview of the ins and outs of mRNA electroporation, discussing the different parameters involved in mRNA electroporation as well as the production of research-grade and production and application of clinical-grade mRNA for gene transfer in the context of cell-based immunotherapies.
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Bertling, Wolf. "Transfection of a DNA/protein complex into nuclei of mammalian cells using polyoma capsids and electroporation". Bioscience Reports 7, n.º 2 (1 de febrero de 1987): 107–12. http://dx.doi.org/10.1007/bf01121873.
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We used empty capsids ofpolyoma virus to transfer DNA fragments and DNA/protein complexes into human cells. We encapsulated labeled and unlabeled single stranded DNA fragments by viral capsids. A complex of DNA with a DNA binding protein, recA, will also be taken up by the capsids, whereas the free protein is not incorporated. We further compared this gentle biological method of DNA transfection with a well-established physical method, electroporation. Electroporation also allows the transfer of DNA as well as protein into cells, although there is no proof that a DNA/protein complex can survive the procedure functionally. Whereas the viability of capsid transfected cells is unaffected (100%), electroporation reduces the viability to 90–95%. On the other hand, the amount of DNA found in the nucleus of electroporated cells is higher than for cells treated with loaded viral capsids.
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Dai, Yang, Yinchang Zhu, Donald A. Harn, Xiaoting Wang, Jianxia Tang, Song Zhao, Fei Lu y Xiaohong Guan. "DNA Vaccination by Electroporation and Boosting with Recombinant Proteins Enhances the Efficacy of DNA Vaccines for Schistosomiasis Japonica". Clinical and Vaccine Immunology 16, n.º 12 (7 de octubre de 2009): 1796–803. http://dx.doi.org/10.1128/cvi.00231-09.
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ABSTRACT Schistosomiasis japonica is an endemic, zoonotic disease of major public health importance in China. Control programs combining chemotherapy and snail killing have not been able to block transmission of infection in lakes and marsh regions. Vaccination is needed as a complementary approach to the ongoing control programs. In the present study, we wanted to determine if the efficacies of DNA vaccines encoding the 23-kDa tetraspanin membrane protein (SjC23), triose phosphate isomerase (SjCTPI), and sixfold-repeated genes of the complementarity determining region 3 (CDR3) in the H chain of NP30 could be enhanced by boosting via electroporation in vivo and/or with cocktail protein vaccines. Mice vaccinated with cocktail DNA vaccines showed a significant worm reduction of 32.88% (P < 0.01) and egg reduction of 36.20% (P < 0.01). Vaccine efficacy was enhanced when animals were boosted with cocktail protein vaccines; adult worm and liver egg burdens were reduced 45.35% and 48.54%, respectively. Nearly identical results were obtained in mice boosted by electroporation in vivo, with adult worm and egg burdens reduced by 45.00% and 50.88%, respectively. The addition of a protein vaccine boost to this regimen further elevated efficacy to approximately 60% for adult worm burden and greater than 60% for liver egg reduction. The levels of interleukin-2, gamma interferon, and the ratios of immunoglobulin G2a (IgG2a)/IgG1 clearly showed that cocktail DNA vaccines induced CD4+ Th1-type responses. Boosting via either electroporation or with recombinant proteins significantly increased associated immune responses over those seen in mice vaccinated solely with DNA vaccines. Thus, schistosome DNA vaccine efficacy was significantly enhanced via boosting by electroporation in vivo and/or cocktail protein vaccines.
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Muriel, Joaquin M., Andrea O’Neill, Jaclyn P. Kerr, Emily Kleinhans-Welte, Richard M. Lovering y Robert J. Bloch. "Keratin 18 is an integral part of the intermediate filament network in murine skeletal muscle". American Journal of Physiology-Cell Physiology 318, n.º 1 (1 de enero de 2020): C215—C224. http://dx.doi.org/10.1152/ajpcell.00279.2019.
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Intermediate filaments (IFs) contribute to force transmission, cellular integrity, and signaling in skeletal muscle. We previously identified keratin 19 (Krt19) as a muscle IF protein. We now report the presence of a second type I muscle keratin, Krt18. Krt18 mRNA levels are about half those for Krt19 and only 1:1,000th those for desmin; the protein was nevertheless detectable in immunoblots. Muscle function, measured by maximal isometric force in vivo, was moderately compromised in Krt18-knockout ( Krt18-KO) or dominant-negative mutant mice ( Krt18 DN), but structure was unaltered. Exogenous Krt18, introduced by electroporation, was localized in a reticulum around the contractile apparatus in wild-type muscle and to a lesser extent in muscle lacking Krt19 or desmin or both proteins. Exogenous Krt19, which was either reticular or aggregated in controls, became reticular more frequently in Krt19-null than in Krt18-null, desmin-null, or double-null muscles. Desmin was assembled into the reticulum normally in all genotypes. Notably, all three IF proteins appeared in overlapping reticular structures. We assessed the effect of Krt18 on susceptibility to injury in vivo by electroporating siRNA into tibialis anterior (TA) muscles of control and Krt19-KO mice and testing 2 wk later. Results showed a 33% strength deficit (reduction in maximal torque after injury) compared with siRNA-treated controls. Conversely, electroporation of siRNA to Krt19 into Krt18-null TA yielded a strength deficit of 18% after injury compared with controls. Our results suggest that Krt18 plays a complementary role to Krt19 in skeletal muscle in both assembling keratin-based filaments and transducing contractile force.
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Lan, Chen-Yu, Ping-Heng Tan, Jiin-Tsuey Cheng, Hsiao-Feng Lu, Ming-Wei Lin, Po-Ni Hsiao y Chung-Ren Lin. "Immunoneutralization of c-Fos Using Intrathecal Antibody Electroporation Attenuates Chronic Constrictive Injury-induced Hyperalgesia and Regulates Preprodynorphin Expression in Rats". Anesthesiology 99, n.º 4 (1 de octubre de 2003): 938–46. http://dx.doi.org/10.1097/00000542-200310000-00029.
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Background In vivo electroporation has been successfully used for the introduction of DNA, RNA, oligonucleotides, and proteins into cells for experimental and therapeutic purposes. The authors evaluated the efficacy of electroporation-mediated c-Fos antibody therapy for neuropathic pain in vitro and in vivo. Methods First, the authors studied the inhibitory effects of intrathecal c-Fos antibody electroporation on the activating protein (AP-1) promoter activity in cultured spinal neuronal cells transfected with p-AP-Luc plasmid and activated with 100 microm glutamate. The inhibitory effect of c-Fos antibody electroporation in the regulation of AP-1 promoter activity was assessed according to the relative luciferase activity. Second, rats with chronic constrictive injury underwent electroporation treatment for neuropathic pain using c-Fos antibody. Thermal nociceptive thresholds were measured before chronic constrictive injury and then on even-numbered days, up to and including day 14, to assess and compare the therapeutic effects of intrathecal electroporation. The time course was assessed by Western blot analysis and by immunohistochemical analysis. Pronociceptive gene expression was measured by assessing prodynorphin mRNA and dynorphin peptides on days 2 and 10 after intrathecal c-Fos electroporation. Results Cotransfection of c-Fos antibody significantly decreased glutamate-induced AP-1 activity. Intrathecal electrotransfer of c-Fos antibody attenuated spinal dynorphin levels, as manifested by significantly elevated pain thresholds in the chronic constrictive injury-affected limbs. Conclusion This study shows that transfer of antibody into rat spinal cords by intrathecal electroporation is a useful method to study the function of endogenous factors of spinal-related disorders.
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Lambert, Helene, Roumen Pankov, Johanne Gauthier y Ronald Hancock. "Electroporation-mediated uptake of proteins into mammalian cells". Biochemistry and Cell Biology 68, n.º 4 (1 de abril de 1990): 729–34. http://dx.doi.org/10.1139/o90-105.
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Proteins of up to 230 kilodaltons are taken up by Chinese hamster ovary fibroblasts exposed to electroporation under conditions generally similar to those used to mediate DNA transfection. The uptake of catalase, ovalbumin, and histone H1 labelled with fluorescein was visualized by fluorescence microscopy. Under the same conditions, the uptake of colloidal gold particles (20 nm diameter) was visualized by electron microscopy. In optimum conditions, about 25% of the cells remained viable and grew normally and about 25% of these retained labelled proteins during two cycles of further growth. About 6 × 104 molecules of catalase were retained per cell. Proteins were taken up when presented to the cells up to 4 h after electroporation, suggesting that mechanisms other than classical electropore formation may operate in these conditions. The proteins were localized in the cytoplasm in a predominantly vesicular pattern and histone H1 entered the nucleus in some cells.Key words: electroporation, protein uptake, gold particle uptake, endocytosis, cell membrane.
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Pham, Tien Thi My y Trang Thi Phuong Phan. "Establishment of electroporation protocol for Bacillus subtilis 1012 and WB800N". Science and Technology Development Journal 18, n.º 2 (30 de junio de 2015): 16–24. http://dx.doi.org/10.32508/stdj.v18i2.1139.
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Bacillus subtilis has been developed as an attractive expression host because of many advantages. For examples, it is nonpathogenic and allows secretion of functional extracellular proteins directly into the culture medium; about 60 % of industrial enzymes available produced by Bacillus species. To use B. subtilis strain for research and as host strain for expression of recombinant protein, bacterial genetic methods should be developed. Electroporation to transfer directly DNA into B. subtilis is one of the methods that draw a lot of attention of scientists. A problem encountered in the methods that draw a lot of attention of scientists. A problem encountered in the electroporation of DNA into B. subtilis is that an established protocol for one strain can hardly be used for another strain. B. subtilis 1012 and WB800N have recently been used as expression hosts for expression of recombinant proteins, but electroporation method has not been established. In this study, we use a pHT plasmid to establish an electroporation protocol for B. subtilis 1012 and WB800N. The influence of sampling time, concentration and time for incubating with lysozyme, voltage on the transformation was investigated to establish the protocol.
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Carpenter, Meredith L. y W. Zacheus Cande. "Using Morpholinos for Gene Knockdown in Giardia intestinalis". Eukaryotic Cell 8, n.º 6 (17 de abril de 2009): 916–19. http://dx.doi.org/10.1128/ec.00041-09.
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ABSTRACT We used translation-blocking morpholinos to reduce protein levels in Giardia intestinalis. Twenty-four hours after electroporation with morpholinos targeting either green fluorescent protein or kinesin-2b, levels of these proteins were reduced by 60%. An epitope-tagged transgene can also be used as a reporter for morpholino efficacy with targets lacking specific antibodies.
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Nunamaker, Elizabeth A., Hai-Ying Zhang, Yuichi Shirasawa, Joseph N. Benoit y David A. Dean. "Electroporation-mediated delivery of catalytic oligodeoxynucleotides for manipulation of vascular gene expression". American Journal of Physiology-Heart and Circulatory Physiology 285, n.º 5 (noviembre de 2003): H2240—H2247. http://dx.doi.org/10.1152/ajpheart.00350.2003.
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The development of inexpensive and effective approaches to transiently decrease gene expression in vivo would be useful for the study of physiological processes in living animals. DNAzymes are a novel class of DNA oligonucleotides that can catalytically cleave target mRNAs and thereby reduce protein production. However, current methods for their delivery in vivo are limited and inefficient. In this study, we show that electroporation can be used to deliver DNAzymes to the intact mesenteric vasculature of rats. With the use of PKC-ϵ as a target, a set of wild-type and mutant control DNAzymes was designed and shown to reduce both PKC-ϵ mRNA and protein levels in cultured smooth muscle cells in a specific manner. The wild-type DNAzyme reduced PKC-ϵ protein levels by 70% at 24 h in two different cell lines without decreasing the levels of the five other PKC isoforms tested. When delivered to the intact vasculature using electroporation, the DNAzyme reduced PKC-ϵ protein levels by >60% without affecting these other PKC isoforms. Electroporation was required for oligonucleotide transfer and was able to deliver the DNAzymes to multiple cell layers in the vessel wall. Protein levels were reduced maximally by 24 h postelectroporation and returned to normal by 48 h. These results suggest that electroporation can be used to deliver DNAzymes and other DNA oligonucleotides to the vasculature in vivo and can decrease gene expression for a window of time that can be used for experimental studies.
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Pollard, Charlotte, Gaëlle Vandermeulen, Guido Vanham, Johan Grooten, Véronique Préat y Stefaan De Koker. "Use of mRNA for vaccination or gene therapy: delivery route determines the verdict (P4316)". Journal of Immunology 190, n.º 1_Supplement (1 de mayo de 2013): 45.6. http://dx.doi.org/10.4049/jimmunol.190.supp.45.6.
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Abstract The use of DNA and viral vector vaccines for the induction of cellular immune responses is increasingly gaining interest. However, concerns have been raised regarding the safety of these immunization strategies. Due to their lack of genome integration, mRNA-based vaccines have emerged as a promising alternative. In this study we evaluated the potency of antigen-encoding mRNA either delivered via complexation with the cationic lipid DOTAP/DOPE, or delivered via in vivo electroporation as a novel vaccination approach. By using luciferase-encoding mRNA we showed that in vivo electroporation of mRNA induces high levels of luciferase at the injection site, while protein expression in mice injected with DOTAP/DOPE complexed mRNA was barely above background levels. Despite the high levels of protein expression, in vivo electroporation of mRNA failed to elicit immune responses against the encoded antigen. In contrast, lipid-based delivery of mRNA induced antigen-specific, functional T cell responses resulting in specific killing of peptide-pulsed cells and the induction of humoral responses. In summary, in vivo electroporation of antigen-encoding mRNA induces high levels of protein expression but no immune responses, thus presenting an ideal tool for mRNA-based gene therapy, while lipid-based delivery offers advantages for vaccination purposes due to the induction of low protein levels but strong antigen-specific cellular immunity.
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Lambert, C. A., P. Y. Lefebvre, B. V. Nusgens y C. M. Lapière. "Modulation of expression of endogenous collagenase and collagen genes by electroporation: possible involvement of Ca2+ and protein kinase C". Biochemical Journal 290, n.º 1 (15 de febrero de 1993): 135–38. http://dx.doi.org/10.1042/bj2900135.
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We have investigated the effect of electroporation on the expression of collagen alpha 1(I), collagenase, c-fos and c-jun genes in human dermal fibroblasts (HDF), human smooth muscle cells (HSMC) and HeLa cells. Collagenase and collagen mRNA levels were respectively increased and decreased in a voltage-dependent manner in HDF harvested 2 days after a sham electroporation. These effects were still observed 10 days after electroporation. Similar effects occurred in electroporated HSMC. Neither collagen nor collagenase mRNAs were detected in control or electroporated HeLa cells. c-fos and c-jun mRNA levels were also increased in electroporated HDF, HSMC and HeLa cells harvested 1 h after plating. This suggests that factor AP1 (fos/jun) could mediate the up-regulation of collagenase expression in electroporated HDF and HSMC. When electroporation of HDF was performed in the presence of H7, an inhibitor of protein kinase C, no increase in collagenase mRNA level was observed, suggesting that protein kinase C might be involved in the transduction of the effect. All the effects reported were also suppressed when cells were electroporated in a medium containing EGTA, suggesting that Ca2+ might mediate the transduction of this effect.
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Sokołowska, Emilia y Agnieszka Urszula Błachnio-Zabielska. "A Critical Review of Electroporation as A Plasmid Delivery System in Mouse Skeletal Muscle". International Journal of Molecular Sciences 20, n.º 11 (6 de junio de 2019): 2776. http://dx.doi.org/10.3390/ijms20112776.
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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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KRAUTZ-PETERSON, GREICE, RITA BHARDWAJ, ZAHRA FAGHIRI, CIBELE A. TARARAM y PATRICK J. SKELLY. "RNA interference in schistosomes: machinery and methodology". Parasitology 137, n.º 3 (21 de septiembre de 2009): 485–95. http://dx.doi.org/10.1017/s0031182009991168.
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SUMMARYRNA interference (RNAi) is a potent gene silencing process that is playing an increasingly important role in investigations of gene function in schistosomes. Here we review what is known about the process in these parasites and provide an update on the methodology and machinery of RNAi. Data are presented to demonstrate that: (1) not all schistosome genes can be suppressed to the same extent, using the methods employed here; (2) while there is variation in the level of suppression achieved for one target gene (SmAP) in adult parasites, all individuals exhibit robust (>80%) suppression; (3) short interfering RNAs (siRNAs) can effect suppression when delivered by soaking (and not just via electroporation, as reported previously); (4) Male/female adult pairs need not be separated prior to siRNA delivery by electroporation for effective gene suppression in both genders and (5) electroporation of siRNAs in medium is as efficient as in commercial electroporation buffer. Regarding the machinery of RNAi in schistosomes, a homologue of the C. elegans multi-membrane spanning, RNA importing protein SID-1 is identified in silico. The gene encoding this protein contains 21 exons and spans over 50 kb to potentially encode a 115,556 Mr protein (SmSID-1). These analyses, and a review of the literature, permit us to derive and present here a draft of potential RNAi pathways in schistosomes.
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HAJDUCH, Eric, J. Carlos ALEDO, Colin WATTS y Harinder S. HUNDAL. "Proteolytic cleavage of cellubrevin and vesicle-associated membrane protein (VAMP) by tetanus toxin does not impair insulin-stimulated glucose transport or GLUT4 translocation in rat adipocytes". Biochemical Journal 321, n.º 1 (1 de enero de 1997): 233–38. http://dx.doi.org/10.1042/bj3210233.
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Acute insulin stimulation of glucose transport in fat and skeletal muscle occurs principally as a result of the hormonal induced translocation of the GLUT4 glucose transporter from intracellular vesicular stores to the plasma membrane. The precise mechanisms governing the fusion of GLUT4 vesicles with the plasma membrane are very poorly understood at present but may share some similarities with synaptic vesicle fusion, as vesicle-associated membrane protein (VAMP) and cellubrevin, two proteins implicated in the process of membrane fusion, are resident in GLUT4-containing vesicles isolated from rat and murine 3T3-L1 adipocytes respectively. In this study we show that proteolysis of both cellubrevin and VAMP, induced by electroporation of isolated rat adipocytes with tetanus toxin, does not impair insulin-stimulated glucose transport or GLUT4 translocation. The hormone was found to stimulate glucose uptake by approx. 16-fold in freshly isolated rat adipocytes. After a single electroporating pulse, the ability of insulin to activate glucose uptake was lowered, but the observed stimulation was nevertheless nearly 5-fold higher than the basal rate of glucose uptake. Electroporation of adipocytes with 600 nM tetanus toxin resulted in a complete loss of both cellubrevin and VAMP expression within 60 min. However, toxin-mediated proteolysis of both these proteins had no effect on the ability of insulin to stimulate glucose transport which was elevated approx. 5-fold, an activation of comparable magnitude to that observed in cells electroporated without tetanus toxin. The lack of any significant change in insulin-stimulated glucose transport was consistent with the finding that toxin-mediated proteolysis of both cellubrevin and VAMP had no detectable effect on insulin-induced translocation of GLUT4 in adipocytes. Our findings indicate that, although cellubrevin and VAMP are resident proteins in adipocyte GLUT4-containing vesicles, they are not required for the acute insulin-induced delivery of GLUT4 to the plasma membrane.
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Hakim, Bilal Ahmad, Vaishali Tyagi, Saurabh Kumar Agnihotri, Amar Nath, Ankit Kumar Agrawal, Ankita Jain, Deependra Singh, Rituraj Konwar y Monika Sachdev. "Electroporation of Mouse Follicles, Oocytes and Embryos without Manipulating Zona Pellucida". Journal of Developmental Biology 9, n.º 2 (1 de abril de 2021): 13. http://dx.doi.org/10.3390/jdb9020013.
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Electroporation is an effective technique of transfection, but its efficiency depends on the optimization of various parameters. In this study, a simplified and efficient method of gene manipulation was standardized through electroporation to introduce a recombinant green fluorescent protein (GFP) construct as well as RNA-inhibitors in intact mouse follicles, oocytes and early embryos, where various electroporation parameters like voltage, pulse number and pulse duration were standardized. Electroporated preantral follicles were cultured further in vitro to obtain mature oocytes and their viability was confirmed through the localization of a known oocyte maturation marker, ovastacin, which appeared to be similar to the in vivo-derived mature oocytes and thus proved the viability of the in vitro matured oocytes after electroporation. Standardized electroporation parameters, i.e., three pulses of 30 V for 1 millisecond at an interval of 10 s, were applied to manipulate the expression of mmu-miR-26a in preantral follicles through the electroporation of miR inhibitors and mimics. The TUNEL apoptosis assay confirmed the normal development of the electroporated embryos when compared to the normal embryos. Conclusively, for the first time, this study demonstrated the delivery of exogenous oligonucleotides into intact mouse follicles, oocytes and embryos without hampering their zona pellucida (ZP) and further development.
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Daud, Adil I., Ronald C. DeConti, Stephanie Andrews, Patricia Urbas, Adam I. Riker, Vernon K. Sondak, Pamela N. Munster et al. "Phase I Trial of Interleukin-12 Plasmid Electroporation in Patients With Metastatic Melanoma". Journal of Clinical Oncology 26, n.º 36 (20 de diciembre de 2008): 5896–903. http://dx.doi.org/10.1200/jco.2007.15.6794.
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Purpose Gene-based immunotherapy for cancer is limited by the lack of safe, efficient, reproducible, and titratable delivery methods. Direct injection of DNA into tissue, although safer than viral vectors, suffers from low gene transfer efficiency. In vivo electroporation, in preclinical models, significantly enhances gene transfer efficiency while retaining the safety advantages of plasmid DNA. Patients and Methods A phase I dose escalation trial of plasmid interleukin (IL)-12 electroporation was carried out in patients with metastatic melanoma. Patients received electroporation on days 1, 5, and 8 during a single 39-day cycle, into metastatic melanoma lesions with six 100-μs pulses at a 1,300-V/cm electric field through a penetrating six-electrode array immediately after DNA injection. Pre- and post-treatment biopsies were obtained at defined time points for detailed histologic evaluation and determination of IL-12 protein levels. Results Twenty-four patients were treated at seven dose levels, with minimal systemic toxicity. Transient pain after electroporation was the major adverse effect. Post-treatment biopsies showed plasmid dose proportional increases in IL-12 protein levels as well as marked tumor necrosis and lymphocytic infiltrate. Two (10%) of 19 patients with nonelectroporated distant lesions and no other systemic therapy showed complete regression of all metastases, whereas eight additional patients (42%) showed disease stabilization or partial response. Conclusion This report describes the first human trial, to our knowledge, of gene transfer utilizing in vivo DNA electroporation. The results indicated this modality to be safe, effective, reproducible, and titratable.
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Campbell, M. A., C. S. Kinlaw y D. B. Neale. "Expression of luciferase and β-glucuronidase in Pinusradiata suspension cells using electroporation and particle bombardment". Canadian Journal of Forest Research 22, n.º 12 (1 de diciembre de 1992): 2014–18. http://dx.doi.org/10.1139/x92-265.
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The reporter gene β-glucuronidase, under the control of the 35S promoter from cauliflower mosaic virus, was introduced into a suspension-cultured cell line of Pinusradiata using electroporation and particle bombardment. Electroporation of suspension-derived protoplasts in the presence of pBI221 resulted in transient β-glucuronidase activity. Maximum levels of β-glucuronidase activity were obtained 24 h after electroporation using field strengths of 1000 V/cm but decreased to background levels within 48 h. This decrease corresponded to a drop in protoplast viability and metabolic activity, as determined by fluorescein diacetate staining and chlorophyll fluorescence. Electroporation of P. radiata protoplasts with a plasmid containing a firefly luciferase reporter gene driven by a 35S promoter resulted in expression levels that were 2–3.5 times that of background. Biolistic transfer of a β-glucuronidase coding sequence driven by a 35S promoter resulted in histochemically detectable β-glucuronidase activity in P. radiata suspension culture cells. Extracts from P. radiata suspension culture cells containing 800 μg soluble protein inhibited the β-glucuronidase activity of Escherichiacoli extracts by 50%. Aliquots of P. radiata extracts containing less than 100 μg of total soluble protein did not exhibit any inhibitory effect. These studies establish two different methods of gene transfer into a P. radiata cell line and will contribute to our ability to examine a variety of promoter types associated with transcriptional regulation in conifers.
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Rosemberg, Y., M. Rotenberg y R. Korenstein. "Electroporation of the photosynthetic membrane: structural changes in protein and lipid-protein domains". Biophysical Journal 67, n.º 3 (septiembre de 1994): 1060–66. http://dx.doi.org/10.1016/s0006-3495(94)80571-5.
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Kapoor, M., C. A. Curle, S. Kalia y Y. Achari. "Minimal promoter for the NAD+-specific glutamate dehydrogenase gene of Neurospora crassa". Biochemistry and Cell Biology 80, n.º 2 (1 de abril de 2002): 177–88. http://dx.doi.org/10.1139/o01-229.
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The expression of the NAD+-specific glutamate dehydrogenase (NAD-GDH) gene of Neurospora crassa is subject to catabolite repression. To identify the minimal sequence necessary for promoter function, the 5'-flanking region of the NAD-GDH gene was screened for potential protein-binding sites. Fragments of DNA, containing sequences upstream from the ATG initiation codon, were employed as probes of Southwestern blots of total cellular protein from cells grown in media promoting repression and induction of NAD-GDH. Two polypeptides interacted differentially with a promoter probe; one was present in greater abundance in repressed cells and a higher relative level of the second was witnessed in induced cells. Electrophoretic mobility shift assays with labeled promoter fragments exhibited preferential interaction with proteins in the induced cultures. The upstream sequence containing the putative protein-binding sites was fused with the coding sequence of the green fluorescent protein (GFP). The resulting plasmid was introduced into the microconidia of an albino mutant of N. crassa by electroporation. Stable integration of the plasmid and expression of GFP in the hyphae and conidia of the transformants were demonstrated by Southern and Western blot analysis and fluorescence microscopy.Key words: Neurospora crassa, repression, induction, GFP fusion, electroporation, microconidia.
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Mukhopadhya, Anindya, Dimitrios Tsiapalis, Niamh McNamee, Brian Talbot y Lorraine O’Driscoll. "Doxorubicin Loading into Milk and Mesenchymal Stem Cells’ Extracellular Vesicles as Drug Delivery Vehicles". Pharmaceutics 15, n.º 3 (21 de febrero de 2023): 718. http://dx.doi.org/10.3390/pharmaceutics15030718.
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Extracellular vesicles (EVs) have great potential as drug delivery vehicles. While mesenchymal/stromal stem cell (MSC) conditioned medium (CM) and milk are potentially safe and scalable sources of EVs for this purpose, the suitability of MSC EVs and milk EVs as drug delivery vehicles has never been compared and so was the objective of this study. Here EVs were separated from MSCs’ CM and from milk and were characterised by nanoparticle tracking analysis, transmission electron microscopy, total protein quantification, and immunoblotting. An anti-cancer chemotherapeutic drug, doxorubicin (Dox), was then loaded into the EVs by one of three methods: by passive loading or by active loading by either electroporation or sonication. Dox-loaded EVs were analysed by fluorescence spectrophotometer, high-performance liquid chromatography (HPLC), and imaging flow cytometer (IFCM). Our study showed that EVs were successfully separated from the milk and MSC CM, with significantly (p < 0.001) higher yields of milk EVs/mL starting material compared to MSC EVs/mL of starting material. Using a fixed amount of EVs for each comparison, electroporation achieved significantly more Dox loading when compared to passive loading (p < 0.01). Indeed, of 250 µg of Dox made available for loading, electroporation resulted in 90.1 ± 12 µg of Dox loading into MSC EVs and 68.0 ± 10 µg of Dox loading into milk EVs, as analysed by HPLC. Interestingly, compared to the passive loading and electroporation approach, after sonication significantly fewer CD9+ EVs/mL (p < 0.001) and CD63+ EVs/mL (p < 0.001) existed, as determined by IFCM. This observation indicates that sonication, in particular, may have detrimental effects on EVs. In conclusion, EVs can be successfully separated from both MSC CM and milk, with milk being a particularly rich source. Of the three methods tested, electroporation appears to be superior for achieving maximum drug loading while not causing damage to EV surface proteins.
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Haruta, Tetsuro, Aaron J. Morris, Peter Vollenweider, James G. Nelson, David W. Rose, Michael Mueckler y Jerrold M. Olefsky. "Ligand-Independent GLUT4 Translocation Induced by Guanosine 5′-O-(3-Thiotriphosphate) Involves Tyrosine Phosphorylation*". Endocrinology 139, n.º 1 (1 de enero de 1998): 358–64. http://dx.doi.org/10.1210/endo.139.1.5698.
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Abstract To delineate the signaling pathway leading to glucose transport protein (GLUT4) translocation, we examined the effect of microinjection of the nonhydrolyzable GTP analog, guanosine 5′-O-(3-thiotriphosphate) (GTPγS), into 3T3-L1 adipocytes. Thirty minutes after the injection of 5 mm GTPγS, 40% of injected cells displayed surface GLUT4 staining indicative of GLUT4 translocation compared with 55% for insulin-treated cells and 10% in control IgG-injected cells. Treatment of the cells with the phosphatidylinositol 3-kinase inhibitor wortmannin or coinjection of GST-p85 SH2 fusion protein had no effect on GTPγS-mediated GLUT4 translocation. On the other hand, coinjection of antiphosphotyrosine antibodies (PY20) blocked GTPγS-induced GLUT4 translocation by 65%. Furthermore, microinjection of GTPγS led to the appearance of tyrosine-phosphorylated proteins around the periphery of the plasma membrane, as observed by immunostaining with PY20. Treatment of the cells with insulin caused a similar phosphotyrosine-staining pattern. Electroporation of GTPγS stimulated 2-deoxy-d-glucose transport to 70% of the extent of insulin stimulation. In addition, immunoblotting with phosphotyrosine antibodies after electroporation of GTPγS revealed increased tyrosine phosphorylation of several proteins, including 70- to 80-kDa and 120- to 130-kDa species. These results suggest that GTPγS acts upon a signaling pathway either downstream of or parallel to activation of phosphatidylinositol 3-kinase and that this pathway involves tyrosine-phosphorylated protein(s).
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Van Tendeloo, Viggo F. I., Peter Ponsaerts, Filip Lardon, Griet Nijs, Marc Lenjou, Christine Van Broeckhoven, Dirk R. Van Bockstaele y Zwi N. Berneman. "Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells". Blood 98, n.º 1 (1 de julio de 2001): 49–56. http://dx.doi.org/10.1182/blood.v98.1.49.
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Abstract Designing effective strategies to load human dendritic cells (DCs) with tumor antigens is a challenging approach for DC-based tumor vaccines. Here, a cytoplasmic expression system based on mRNA electroporation to efficiently introduce tumor antigens into DCs is described. Preliminary experiments in K562 cells using an enhanced green fluorescent protein (EGFP) reporter gene revealed that mRNA electroporation as compared with plasmid DNA electroporation showed a markedly improved transfection efficiency (89% versus 40% EGFP+ cells, respectively) and induced a strikingly lower cell toxicity (15% death rate with mRNA versus 51% with plasmid DNA). Next, mRNA electroporation was applied for nonviral transfection of different types of human DCs, including monocyte-derived DCs (Mo-DCs), CD34+ progenitor-derived DCs (34-DCs) and Langerhans cells (34-LCs). High-level transgene expression by mRNA electroporation was obtained in more than 50% of all DC types. mRNA-electroporated DCs retained their phenotype and maturational potential. Importantly, DCs electroporated with mRNA-encoding Melan-A strongly activated a Melan-A–specific cytotoxic T lymphocyte (CTL) clone in an HLA-restricted manner and were superior to mRNA-lipofected or -pulsed DCs. Optimal stimulation of the CTL occurred when Mo-DCs underwent maturation following mRNA transfection. Strikingly, a nonspecific stimulation of CTL was observed when DCs were transfected with plasmid DNA. The data clearly demonstrate that Mo-DCs electroporated with mRNA efficiently present functional antigenic peptides to cytotoxic T cells. Therefore, electroporation of mRNA-encoding tumor antigens is a powerful technique to charge human dendritic cells with tumor antigens and could serve applications in future DC-based tumor vaccines.
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Verhoef, K., S. E. C. Koken y B. Berkhout. "Electroporation of the HIV Tat trans-Activator Protein into Cells". Analytical Biochemistry 210, n.º 1 (abril de 1993): 210–14. http://dx.doi.org/10.1006/abio.1993.1176.
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Semenova, Nina P., Katarina Znidar, Masa Bosnjak, Maja Cemazar y Loree C. Heller. "Nucleic acid sensing in the cytoplasm of C2C12 cells directly after plasmid DNA electroporation". Journal of Immunology 200, n.º 1_Supplement (1 de mayo de 2018): 169.12. http://dx.doi.org/10.4049/jimmunol.200.supp.169.12.
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Abstract Mounting evidence stresses the importance of nucleic acid sensing in the cytoplasm of living cells in a variety of physiological and pathological processes, including anti-pathogen defense, autoimmunity and development of malignant tumors. A subset of pattern-recognition receptors (PRRs) are nucleic acid sensors widely expressed in a variety of cells, but the detailed mechanisms of nucleic acid sensing have not been clarified. Our previous studies confirmed the cell type-specific upregulation of PRRs on both the mRNA and protein levels after electroporation of plasmid DNA (pDNA) into the cells. Type I interferon was found to be upregulated as well, which implicates innate immunity pathway activation in response to pDNA entry into the cytoplasm. Still, the sequence of events after pDNA electroporation remains unclear. Using molecular and proteomic techniques, we identified DAI/ZBP1 protein as the initial responding PRR to pDNA entering the cytoplasm after electroporation and estimated the participation of other PRRs in this process in mouse myoblast C2C12 cells. Better understanding of molecular determinants of nucleic acid sensing by PRRs may allow us to manipulate this process, developing new and improving existing therapies for immunity- and tumor-related diseases.
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Dewi, Raden Roro Sri Pudji Sinarni, Alimuddin Alimuddin, Agus Oman Sudrajat, Komar Sumantadinata y Sularto Sularto. "OPTIMAL ELECTROPORATION CONDITION FOR SPERM MEDIATED GENE TRANSFER IN STRIPPED CATFISH (Pangasionodon hypophthalmus)". Indonesian Aquaculture Journal 5, n.º 1 (30 de junio de 2010): 1. http://dx.doi.org/10.15578/iaj.5.1.2010.1-10.
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The success of transgenic fish production has been achieved through eggs fertilization using electroporated sperms carrying exogenous DNA. This study was conducted in order to obtain the optimal electroporation condition for stripped catfish sperm. A plasmid containing green fluorescent protein (GFP) gene driven by carp β-actin promoter was transferred into sperm using electrophoresis method towards transgenic stripped catfish (Pangasionodon hypophthalmus) production. Electroporation was carried out using square wave shock with pulse length of 30 ms and pulse interval of 0.1 sec. Treatments are combination between voltage (50 V, 75 V, and 100 V) and pulse number (1 and 3). Exogenous DNA concentration used was 10 μg/mL of Tris-EDTA. Results showed that increasing the voltage from 50 to 100 decreased sperm motility, while pulse number did not affect sperm motility. Voltage of 50 gave the best motility of sperm, although sperm viability relatively similar between treatments and control except at 100 V with 3 pulses number. Further, electroporation-treated sperms were able to fertilize eggs. Higher hatching rate of eggs was obtained in electroporation treatment at 50 V with pulse number of 1 and 3. The persistence of transferred GFP was detected in electroporated and incubated sperms (control). However, GFP was only detected in larvae from eggs that were fertilized by electroporated sperm. Thus, electroporation could be applied to produce transgenic stripped catfish.
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Igawa, Kazunari, Naoko Ohara, Atsushi Kawakubo, Kouji Sugimoto, Kajiro Yanagiguchi, Takeshi Ikeda, Shizuka Yamada y Yoshihiko Hayashi. "D-Glucosamine Promotes Transfection Efficiency during Electroporation". BioMed Research International 2014 (2014): 1–4. http://dx.doi.org/10.1155/2014/485867.
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D-Glucosamine is a useful medicament in various fields of medicine and dentistry. With respect to stability of the cell membrane, it has been reported that bradykinin-induced nociceptive responses are significantly suppressed by the direct application of D-glucosamine. Electroporation is usually used to effectively introduce foreign genes into tissue culture cells. Buffers for electroporation with or without D-glucosamine are used in experiments of transfection vectors. This is the first study to indirectly observe the stability and protection of the osteoblast membrane against both electric stress and gene uptake (the proton sponge hypothesis: osmotic rupture during endosomes prior to fusion with lysosomes) in electroporation with D-glucosamine application. The transfection efficiency was evaluated as the fluorescence intensity of the transfected green fluorescent protein (GFP) in the cultured cells (osteoblasts; NOS-1 cells). The transfection efficiency increased over 30% in the electroporation samples treated with D-glucosamine-supplemented buffer after one day. The membrane absorption of D-glucosamine is the primary mechanism of membrane stress induced by electric stress. This new function of D-glucosamine is useful and meaningful for developing more effective transformation procedures.
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Grasseschi, Helen A. y Michael F. Minnick. "Transformation of Bartonella bacilliformis by electroporation". Canadian Journal of Microbiology 40, n.º 9 (1 de septiembre de 1994): 782–86. http://dx.doi.org/10.1139/m94-123.
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Bartonella bacilliformis is a member of the order Rickettsiales, family Bartonellaceae. The bacterium is an intracellular parasite of human erythrocytes. To date, members of the family Bartonellaceae have not been transformed by standard chemical methods. We report the first successful transformation of a member of the Bartonellaceae family, B. bacilliformis, by the method of electroporation. The optimal conditions for electroporation of B. bacilliformis include a field strength of 12.5 kV/cm and a time constant of 5 ms using 0.2-cm cuvettes. With these parameters and the cosmid pEST (RK2 replicon), a transformation efficiency of 7.8 × 105 was obtained. Transformants were readily cultured on medium containing kanamycin sulfate at concentrations ranging from 15 to 600 μg/mL. Bacterial survival was approximately 31% under optimal electroporation conditions, and the maximal number of transformants was obtained with 80 ng of pEST DNA. Bartonella bacilliformis was verified as the transformed organism by a comparison of transformant protein profiles with those of wild-type B. bacilliformis using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and detection of the exogenous plasmid in DNA from the transformed bacteria by DNA hybridization. Transformations using the plasmids pMK20, pML31, and pUCK18 (containing the replicons ColE1, F, and pMB1, respectively) were not successful.Key words: transformation, electroporation, Rickettsiales order.
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Busch, Christian, Joachim Orth, Nabil Djouder y Klaus Aktories. "Biological Activity of a C-Terminal Fragment ofPasteurella multocida Toxin". Infection and Immunity 69, n.º 6 (1 de junio de 2001): 3628–34. http://dx.doi.org/10.1128/iai.69.6.3628-3634.2001.
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ABSTRACT The protein toxin of Pasteurella multocida PMT is a potent mitogen and activator of phospholipase Cβ. In this study different toxin fragments were investigated. A C-terminal fragment encompassing amino acids 581 through 1285 (PMT581C) was constructed, which was inactive toward intact embryonic bovine lung (EBL) cells after addition to culture medium but caused reorganization of the actin cytoskeleton and rounding up of cells when introduced into the cells by electroporation. As the holotoxin, the toxin fragment PMT581C induced an increase in total inositol phosphate levels after introduction into the cell by electroporation. A C-terminal fragment shorter than PMT581C as well as N-terminal fragments were inactive. Exchange of cysteine-1165 for serine in the holotoxin resulted in a complete loss of the ability to increase inositol phosphate levels. Correspondingly, the mutated toxin fragment PMT581C.C1165S was inactive after cell introduction by electroporation, suggesting an essential role of Cys-1165 in the biological activity of the toxin.
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Kisakov, Denis N., Lyubov A. Orlova, Сергей V. Sharabrin, Andrey P. Rudometov, Maria B. Borgoyakova, Ekaterina V. Starostina, Larisa I. Karpenko y Alexander A. Ilyichev. "Delivery of a DNA vaccine encoding SARS-CoV-2 receptor-binding domain (RBD) by electroporation". Medical academic journal 2, n.º 2 (6 de noviembre de 2022): 191–96. http://dx.doi.org/10.17816/maj108614.
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BACKGROUND: Nucleic acid-based prevention tools provide a promising platform for developing vaccines, including those against COVID-19. Previously, we developed the pVAXrbd DNA vaccine encoding the receptor-binding domain (RBD) of SARS-CoV-2, which, when administered intramuscularly to animals, induced a relatively weak immune response. The next stage of the study is to increase the immune response, in particular, using electroporation as one of the methods for increasing the immunogenicity of DNA vaccines.
AIM: The aim of this article is to evaluate the immune response using electroporation in mice after immunization with pVAXrbd.
MATERIALS AND METHODS: BALB/c mice were immunized with pVAXrbd using direct and reverse polarity square wave direct current electroporation with three pulses of 12 V for 30 ms and an interval of 950 ml with a current limit of 45 mA.
RESULTS: BALB/c mice were immunized twice with an interval of three weeks with a dose of 100 g of DNA. Using ELISA, the titers of RBD-specific antibodies in the group of animals immunized with pVAXrbd using electroporation were 1:109350, which is 16 times higher than in the group of animals that received the DNA vaccine only intramuscularly (titers 1:6750). IFN ELISpot analysis showed that the largest number of cells (2434 spots/splenocytes, million) producing IFN in response to stimulation with peptides from the RBD protein was registered in the group of animals immunized with pVAXrbd using electroporation. For comparison, in the control group, the number of cells is 6.5 times lower: 380 spots / splenocytes, mln.
CONCLUSIONS: Administration of the pVAXrbd DNA vaccine to laboratory animals by electroporation significantly enhances both the humoral and cellular specific immune response compared to intramuscular administration of the naked DNA vaccine.
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DiTommaso, Tia, Julie M. Cole, Luke Cassereau, Joshua A. Buggé, Jacquelyn L. Sikora Hanson, Devin T. Bridgen, Brittany D. Stokes et al. "Cell engineering with microfluidic squeezing preserves functionality of primary immune cells in vivo". Proceedings of the National Academy of Sciences 115, n.º 46 (31 de octubre de 2018): E10907—E10914. http://dx.doi.org/10.1073/pnas.1809671115.
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The translational potential of cell-based therapies is often limited by complications related to effectively engineering and manufacturing functional cells. While the use of electroporation is widespread, the impact of electroporation on cell state and function has yet to be fully characterized. Here, we use a genome-wide approach to study optimized electroporation treatment and identify striking disruptions in the expression profiles of key functional transcripts of human T cells. These genetic disruptions result in concomitant perturbation of cytokine secretion including a 648-fold increase in IL-2 secretion (P < 0.01) and a 30-fold increase in IFN-γ secretion (P < 0.05). Ultimately, the effects at the transcript and protein level resulted in functional deficiencies in vivo, with electroporated T cells failing to demonstrate sustained antigen-specific effector responses when subjected to immunological challenge. In contrast, cells subjected to a mechanical membrane disruption-based delivery mechanism, cell squeezing, had minimal aberrant transcriptional responses [0% of filtered genes misregulated, false discovery rate (FDR) q < 0.1] relative to electroporation (17% of genes misregulated, FDR q < 0.1) and showed undiminished effector responses, homing capabilities, and therapeutic potential in vivo. In a direct comparison of functionality, T cells edited for PD-1 via electroporation failed to distinguish from untreated controls in a therapeutic tumor model, while T cells edited with similar efficiency via cell squeezing demonstrated the expected tumor-killing advantage. This work demonstrates that the delivery mechanism used to insert biomolecules affects functionality and warrants further study.
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He, Gen, Chengduan Yang, Tian Hang, Di Liu, Hui-Jiuan Chen, Ai-hua Zhang, Dian Lin, Jiangming Wu, Bo-ru Yang y Xi Xie. "Hollow Nanoneedle-Electroporation System To Extract Intracellular Protein Repetitively and Nondestructively". ACS Sensors 3, n.º 9 (27 de agosto de 2018): 1675–82. http://dx.doi.org/10.1021/acssensors.8b00367.
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Tanihara, Fuminori, Tatsuya Takemoto, Eri Kitagawa, Shengbin Rao, Lanh Thi Kim Do, Akira Onishi, Yukiko Yamashita et al. "Somatic cell reprogramming-free generation of genetically modified pigs". Science Advances 2, n.º 9 (septiembre de 2016): e1600803. http://dx.doi.org/10.1126/sciadv.1600803.
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Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential for facilitating the genetic modification of pigs.
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Aghvami, Tehrani Azadeh, Saeid Latifi-Navid, Saber Zahri y Mohsen Sagha. "Optimization of pCMV3-Nog-C-GFPSpark Electro-transfection in MCF-7, a Breast Cancer Cell Line". Research Journal of Biotechnology 16, n.º 10 (25 de septiembre de 2021): 13–18. http://dx.doi.org/10.25303/1610rjbt1318.
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Bone morphogenetic protein (BMP) signaling is known as one of the most important pathways in breast cancer tumorigenesis. This triggers the epithelial to mesenchymal transition (EMT) in metastatic cells and consequently the migrating cells become invasive and noggin, a BMP antagonist prevents it. So, the present study aimed to optimize Noggin transfection into MCF-7 as a breast cancer cell line. Following DH5α bacterial cell culturing and pCMV3- Nog-GFPSpark transformation, the resulted plasmid was extracted, purified and finally transfected into MCF-7 at different voltages (100-230V), resistances (1000 and ∞ Ω) and capacitances (25-75μF) using the electroporation system with various concentrations of plasmid (between 30 and 100μg/ml). As a result, we found that noggin has a better transfection into MCF- 7 in 230V, 50μF, 1000 Ωof electroporator. At 80μg/ml concentration of plasmid, the cell expressing GFP also represented the noggin expression.
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Tannig, Pierre, Antonia Sophia Peter, Dennis Lapuente, Stephan Klessing, Dominik Damm, Matthias Tenbusch, Klaus Überla y Vladimir Temchura. "Modulation of Vaccine-Induced HIV-1-Specific Immune Responses by Co-Electroporation of PD-L1 Encoding DNA". Vaccines 8, n.º 1 (14 de enero de 2020): 27. http://dx.doi.org/10.3390/vaccines8010027.
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The importance of a balanced TH1/TH2 humoral immune response against the HIV-1 envelope protein (Env) for antibody-mediated HIV-1 control is increasingly recognized. However, there is no defined vaccination strategy to raise it. Since immune checkpoints are involved in the induction of adoptive immunity and their inhibitors (monoclonal antibodies) are licensed for cancer therapy, we investigated the effect of checkpoint blockade after HIV-1 genetic vaccination on enhancement and modulation of antiviral antibody responses. By intraperitoneal administration of checkpoint antibodies in mice we observed an induction of anti-drug antibodies which may interfere with immunomodulation by checkpoint inhibitors. Therefore, we blocked immune checkpoints locally by co-electroporation of DNA vaccines encoding the active soluble ectodomains of programmed cell death protein-1 (PD-1) or its ligand (PD-L1), respectively. Plasmid-encoded immune checkpoints did not elicit a detectable antibody response, suggesting no interference with their immunomodulatory effects. Co-electroporation of a HIV-1 DNA vaccine formulation with soluble PD-L1 ectodomain increased HIV-1 Env-specific TH1 CD4 T cell and IgG2a antibody responses. The overall antibody response was hereby shifted towards a more TH1/TH2 balanced subtype pattern. These findings indicate that co-electroporation of soluble checkpoint ectodomains together with DNA-based vaccines has modulatory effects on vaccine-induced immune responses that could improve vaccine efficacies.
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Benzoni, E., M. L. Torre, M. Faustini, S. Stacchezzini, F. Cremonesi, U. Conte, S. Villani, V. Russo, G. Ricevuti y D. Vigo. "Transient Transfection of Porcine Granulosa Cells after 3D Culture in Barium Alginate Capsules". International Journal of Immunopathology and Pharmacology 18, n.º 4 (octubre de 2005): 677–81. http://dx.doi.org/10.1177/039463200501800409.
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Three-dimensional culture systems in barium alginate capsules can be employed to maintain primary granulosa cells in an undifferentiated state for almost 6 days. This is due to a self-organization of cells in a pseudofollicular structure. The transfection of primary granulosa cells is a necessary condition when employing these culture systems for several purposes, for example as an in vitro toxicity test or the development of oocytes or zygotes. In this work, the feasibility of two transient transfection techniques (liposome-mediated and electroporation) was assessed in primary porcine granulosa cells after a 6-day culture in an artificial extracellular matrix (barium alginate membrane). Human recombinant green fluorescent protein was chosen as a molecular readout, and protein expression was assessed after 48 hours from transfection. Liposome-mediated transfection gave low transfection levels, with increasing yields from 2 to 12 μgDNA/ml of medium; the maximum percentage (85.7%) was reached at 12 μgDNA/ml of medium. Electroporation-mediated transfection yields were higher: the best results (81.7% of transfected cells) were achieved with two 50V pulses and 12 μg/ml DNA. The application of a single or double pulse (50V) at 4 mgDNA/ml gave negligible results. These results indicate that primary granulosa cell cultured in barium alginate capsules can be transfected by electroporation with high transfection yields.
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Schieffer, B., H. Drexler, B. N. Ling y M. B. Marrero. "G protein-coupled receptors control vascular smooth muscle cell proliferation via pp60c-src and p21ras". American Journal of Physiology-Cell Physiology 272, n.º 6 (1 de junio de 1997): C2019—C2030. http://dx.doi.org/10.1152/ajpcell.1997.272.6.c2019.
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The binding of vasoactive peptides to their respective G protein-coupled receptors has been implicated in the pathogenesis of vascular smooth muscle cell proliferation, leading to the development of hypertension, arteriosclerosis, and restenosis after vascular injury. We previously showed that the cytosolic tyrosine kinase pp60c-src is crucial for angiotensin II (ANG II)-induced activation of the protooncogene p21ras. Therefore, we investigated the role of pp60c-src and p21ras in rat aortic smooth muscle cell proliferation induced by several G protein-coupled receptors. ANG II, endothelin-1, or thrombin increased cell proliferation and DNA synthesis. Electroporation of anti-pp60c-src antibodies into cells abolished proliferation in response to these G protein-coupled receptor ligands but not in response to platelet-derived growth factor-BB (PDGF-BB). In contrast, electroporation of anti-p21ras antibody completely blocked DNA synthesis and cell proliferation in response to ANG II, endothelin-1, thrombin, and PDGF-BB. Our data indicate that the pp60c-src tyrosine kinase is necessary and specific for vascular smooth muscle cell proliferation and DNA synthesis in response to G protein-coupled receptors but not classic growth factor receptors.
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Li, Lin-Hong, Rama Shivakumar, Stephanie Feller, Cornell Allen, Jonathan M. Weiss, Sergey Dzekunov, Vin Singh, John Holaday, Joseph Fratantoni y Linda N. Liu. "Highly Efficient, Large Volume Flow Electroporation". Technology in Cancer Research & Treatment 1, n.º 5 (octubre de 2002): 341–49. http://dx.doi.org/10.1177/153303460200100504.
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Electroporation is widely used to transfect and load cells with various molecules. Traditional electroporation using a static mode is typically restricted to volumes less than 1 mL, which limits its use in clinical and industrial bioprocessing applications. Here we report efficient, large volume transfection results by using a scalable-volume electroporation system. Suspended (Jurkat) and adherent cells (10T1/2 and Huh-7) were tested. A large macromolecule, FITC-conjugated dextran (MW=500 kD) was used to measure cell uptake, while a plasmid carrying the gene coding for enhanced green fluorescence protein (eGFP) was used to quantitate the flow electrotransfection efficiency as determined by flow cytometry. The flow electroloading efficiency of FITC-dextran was >90%, while the cell viability was highly maintained (>90%). High flow electrotransfection efficiency (up to 75%) and cell viability (up to 90%) were obtained with processing volumes ranging from 1.5 to 50 mL. No significant difference of electrotransfection efficiency was observed between flow and static electrotransfection. When 50 mL of cell volume was processed and samples collected at different time points during electroporation, the transgene expression and cell viability results were identical. We also demonstrated that DNA plasmid containing EBNA1-OriP elements from Epstein-Barr virus were more efficient in transgene expression than standard plasmid without the elements (at least 500 too 1000-fold increase in expression level). Finally, to examine the feasibility of utilizing flow electrotransfected cells as a gene delivery vehicle, 10T1/2 cells were transfected with a DNA plasmid containing the gene coding for mIL12. mIL12 transfected cells were injected subcutaneously into mice, and produced functional mIL12, as demonstrated by anti-angiogenic activity. This is the first demonstration of efficient, large volume, flow electroporation and the in vivo efficacy of flow electrotransfected cells. This technology may be useful for clinical gene therapy and large-scale bioprocesses.
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Barnett, Rebecca C., Xin Lin, Michael Barravecchia, Rosemary A. Norman, Karen L. de Mesy Bentley, Fabeha Fazal, Jennifer L. Young y David A. Dean. "Featured Article: Electroporation-mediated gene delivery of surfactant protein B (SP-B) restores expression and improves survival in mouse model of SP-B deficiency". Experimental Biology and Medicine 242, n.º 13 (5 de junio de 2017): 1345–54. http://dx.doi.org/10.1177/1535370217713000.
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Surfactant Protein B Deficiency is a rare but lethal monogenetic, congenital lung disease of the neonate that is unresponsive to any treatment except lung transplantation. Based on the potential that gene therapy offers to treat such intractable diseases, our objective was to test whether an electroporation-based gene delivery approach could restore surfactant protein B expression and improve survival in a compound knockout mouse model of surfactant protein B deficiency. Surfactant protein B expression can be shut off in these mice upon withdrawl of doxycycline, resulting in decreased levels of surfactant protein B within four days and death due to lung dysfunction within four to seven days. Control or one of several different human surfactant protein B-expressing plasmids was delivered to the lung by aspiration and electroporation at the time of doxycycline removal or four days later. Plasmids expressing human surfactant protein B from either the UbC or CMV promoter expressed surfactant protein B in these transgenic mice at times when endogenous surfactant protein B expression was silenced. Mean survival was increased 2- to 5-fold following treatment with the UbC or CMV promoter-driven plasmids, respectively. Histology of all surfactant protein B treated groups exhibited fewer neutrophils and less alveolar wall thickening compared to the control groups, and electron microscopy revealed that gene transfer of surfactant protein B resulted in lamellar bodies that were similar in the presence of electron-dense, concentric material to those in surfactant protein B-expressing mice. Taken together, our results show that electroporation-mediated gene delivery of surfactant protein B-expressing plasmids improves survival, lung function, and lung histology in a mouse model of surfactant protein B deficiency and suggest that this may be a useful approach for the treatment of this otherwise deadly disease. Impact statement Surfactant protein B (SP-B) deficiency is a rare but lethal genetic disease of neonates that results in severe respiratory distress with no available treatments other than lung transplantation. The present study describes a novel treatment for this disease by transferring the SP-B gene to the lungs using electric fields in a mouse model. The procedure is safe and results in enough expression of exogenous SP-B to improve lung histology, lamellar body structure, and survival. If extended to humans, this approach could be used to bridge the time between diagnosis and lung transplantation and could greatly increase the likelihood of affected neonates surviving to transplantation and beyond.
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Zhang, Yuanjun, Zishen Yan, Xingyu Xia y Yuan Lin. "A Novel Electroporation System for Living Cell Staining and Membrane Dynamics Interrogation". Micromachines 11, n.º 8 (11 de agosto de 2020): 767. http://dx.doi.org/10.3390/mi11080767.
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A novel electroporation system was developed to introduce transient membrane pores to cells in a spatially and temporally controlled manner, allowing us to achieve fast electrotransfection and live cell staining as well as to systematically interrogate the dynamics of the cell membrane. Specifically, using this platform, we showed that both reversible and irreversible electroporation could be induced in the cell population, with nano-sized membrane pores in the former case being able to self-reseal in ~10 min. In addition, green fluorescent protein(GFP)-vinculin plasmid and 543 phalloidin have been delivered successively into fibroblast cells, which enables us to monitor the distinct roles of vinculin and F-actin in cell adhesion and migration as well as their possible interplay during these processes. Compared to conventional bulk electroporation and staining methods, the new system offers advantages such as low-voltage operation, cellular level manipulation and testing, fast and adjustable transfection/staining and real-time monitoring; the new system therefore could be useful in different biophysical studies in the future.
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Busatto, Sara, Dalila Iannotta, Sierra A. Walker, Luisa Di Marzio y Joy Wolfram. "A Simple and Quick Method for Loading Proteins in Extracellular Vesicles". Pharmaceuticals 14, n.º 4 (13 de abril de 2021): 356. http://dx.doi.org/10.3390/ph14040356.
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Extracellular vesicles (EVs) mediate intercellular transport of biomolecular cargo in the body, making them promising delivery vehicles for bioactive compounds. Genetic engineering of producer cells has enabled encapsulation of therapeutic proteins in EVs. However, genetic engineering approaches can be expensive, time-consuming, and incompatible with certain EV sources, such as human plasma and bovine milk. The goal of this study was to develop a quick, versatile, and simple method for loading proteins in EVs post-isolation. Proteins, including CRISPR associated protein 9 (Cas9), were bound to cationic lipids that were further complexed with MDA-MB-231 cell-derived EVs through passive incubation. Size-exclusion chromatography was used to remove components that were not complexed with EVs. The ability of EVs to mediate intracellular delivery of proteins was compared to conventional methods, such as electroporation and commercial protein transfection reagents. The results indicate that EVs retain native features following protein-loading and obtain similar levels of intracellular protein delivery as conventional methods, but display less toxicity. This method opens up opportunities for rapid exploration of EVs for protein delivery.
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Lanjewar, S. N. y K. R. Bondioli. "205 Optimization of Transfection Efficiency for CRISPR/Cas9-Induced Genomic Editing in Porcine Fibroblast Cells". Reproduction, Fertility and Development 30, n.º 1 (2018): 243. http://dx.doi.org/10.1071/rdv30n1ab205.
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The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system creates DNA double-stranded breaks (DSB) at specific sequences and allows efficient genomic modification, even in species previously resistant to gene editing. The DSB can be repaired using non-homologous end joining (creating insertions/deletions) or by homology directed repair (HDR) using a donor DNA with small changes at the cut site, giving rise to precise targeted modifications. Despite growing interest in genome editing using RNA-guided endonucleases, the efficiency of HDR is only 0.5 to 20%. The objective of this study was to optimize transfection conditions in order to increase efficiency of HDR for CRISPR/Cas9 targeted genomic editing of porcine cells. We utilised the Swedish mutation of the porcine APP gene causing early-onset Alzheimer’s disease. We first tested co-transfection of 2 plasmids, one containing our guide RNA (gRNA) and another containing the Cas9 nuclease, using square-wave electroporation. Upon analysis via T7 endonuclease assay I, this method failed to produce a DNA DSB at the target site. Next, we tested transfection of a single vector containing both the gRNA and Cas9 nuclease. Three gRNAs targeting exon 17 of the porcine APP gene were constructed and inserted into CRISPR/Cas9 pGuide-it plasmids expressing green fluorescent protein (GFP). Plasmid DNA was transfected into cultured porcine fibroblast cells by 3 methods: Lipofectamine 2000, square-wave electroporation, and exponential-wave electroporation. To determine which method yielded the highest transfection rates, cells were analysed using flow cytometry to detect GFP expression. The transfection efficiency, percentage of cells expressing GFP, was analysed by one-way ANOVA and individual pair wise comparisons. Twelve microliters of Lipofectamine 2000 per well of a 6-well plate with 200 ng of plasmid DNA per μL of Lipofectamine was used to optimize transfection rates, as suggested by the manufacturer. Removal of transfection media after 48 h yielded higher transfection rates than removal after 24 h (6.9% ± 0.7 v. 2.2% ± 0.1; P = 0.02). For electroporation, 12.5 and 25 μg of plasmid DNA per mL was added to 0.2- and 0.4-mm gap cuvettes, respectively, each containing cell suspensions of 1 × 106 cells mL−1. Square-wave electroporation was performed at 300 V for three 1-ms pulses in 0.2-mm cuvettes. Exponential-wave electroporation was performed at 350 V and 500 μFD in both 0.2-mm and 0.4-mm cuvettes. Exponential-wave electroporation containing 25 μg of plasmid DNA/mL of cell suspension yielded the highest average transfection efficiency, 22.8% (P < 0.00001), compared with square-wave electroporation and transfection using optimized Lipofectamine 2000 conditions (9.1 and 1%, respectively). All 3 gRNAs resulted in similar transfection rates. In conclusion, efficiency of transfection of the CRISPR/Cas9 system into porcine cells is optimized using exponential-wave electroporation of a single plasmid CRISPR system.
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Akaneya, Yukio, Bin Jiang y Tadaharu Tsumoto. "RNAi-Induced Gene Silencing by Local Electroporation in Targeting Brain Region". Journal of Neurophysiology 93, n.º 1 (enero de 2005): 594–602. http://dx.doi.org/10.1152/jn.00161.2004.
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Genetic manipulation for “knockout” (KO) is a useful tool for characterizing a target gene. However, its shortcomings that need to be overcome hinder its easy and ready usage in ordinary laboratories. Here we describe a knockdown technique termed the RNA interference (RNAi)-induced gene silencing by local electroporation (RISLE). Small interfering RNA (siRNA) introduction by electroporation into a specific brain region results in a marked reduction in the expression levels of both the mRNA and protein of the target genes such as GluR2 and Cox-1 without affecting the expression levels of proteins other than that of the target protein or causing pathological changes in the target tissues. The effective electrical pulses are relatively weak, consisting of a strong short pulse and a weak long pulse applied in tandem. RISLE can knock down a gene at the target region, for example, the visual cortex and the CA1 region of the hippocampus, without affecting other regions. Moreover, the knockdown models constructed using this technique have physiological functions consistent with previous findings, that is, glutamate release from presynaptic sites, long-term potentiation (LTP), and long-term depression (LTD). These results suggest that this technique is applicable and characterized by spatial flexibility, temporal accessibility, and ease of establishment of knockdown models. The intactness of the tissue subjected to RISLE is due to the weak electrical pulses applied and the limited area of gene silencing. Thus RISLE may be applicable to disease therapy in the future.
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Fujii, Nobuharu, Marni D. Boppart, Scott D. Dufresne, Patricia F. Crowley, Alison C. Jozsi, Kei Sakamoto, Haiyan Yu et al. "Overexpression or ablation of JNK in skeletal muscle has no effect on glycogen synthase activity". American Journal of Physiology-Cell Physiology 287, n.º 1 (julio de 2004): C200—C208. http://dx.doi.org/10.1152/ajpcell.00415.2003.
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c-Jun NH2-terminal kinase (JNK) is highly expressed in skeletal muscle and is robustly activated in response to muscle contraction. Little is known about the biological functions of JNK signaling in terminally differentiated muscle cells, although this protein has been proposed to regulate insulin-stimulated glycogen synthase activity in mouse skeletal muscle. To determine whether JNK signaling regulates contraction-stimulated glycogen synthase activation, we applied an electroporation technique to induce JNK overexpression (O/E) in mouse skeletal muscle. Ten days after electroporation, in situ muscle contraction increased JNK activity 2.6-fold in control muscles and 15-fold in the JNK O/E muscles. Despite the enormous activation of JNK activity in JNK O/E muscles, contraction resulted in similar increases in glycogen synthase activity in control and JNK O/E muscles. Consistent with these findings, basal and contraction-induced glycogen synthase activity was normal in muscles of both JNK1- and JNK2-deficient mice. JNK overexpression in muscle resulted in significant alterations in the basal phosphorylation state of several signaling proteins, such as extracellular signal-regulated kinase 1/2, p90 S6 kinase, glycogen synthase kinase 3, protein kinase B/Akt, and p70 S6 kinase, in the absence of changes in the expression of these proteins. These data suggest that JNK signaling regulates the phosphorylation state of several kinases in skeletal muscle. JNK activation is unlikely to be the major mechanism by which contractile activity increases glycogen synthase activity in skeletal muscle.
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Boll, Antje y Michael Schrader. "Elongation of Peroxisomes as an Indicator for Efficient Dynamin-like Protein 1 Knock Down in Mammalian Cells". Journal of Histochemistry & Cytochemistry 53, n.º 8 (agosto de 2005): 1037–40. http://dx.doi.org/10.1369/jhc.5b6681.2005.
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RNA interference has become a valuable tool to identify and investigate proteins involved in the formation of peroxisomes. We demonstrate that the elongation of peroxisomes serves as an excellent indicator for efficient knock down of dynamin-like protein 1 (DLP1) in mammalian cells. We took advantage of the silencing-dependent morphological changes of peroxisomes to compare different transfection methods and show that a single transfection of DLP1 siRNA by electroporation is sufficient to effectively silence DLP1. We present a fast, easy, and convenient protocol for efficient gene silencing in a large number of cells, which can be used for quantitative and biochemical studies.
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Zeng, Fanning, Valerie Beck, Sven Schuierer, Isabelle Garnier, Carole Manneville, Claudia Agarinis, Lapo Morelli et al. "A Simple and Efficient CRISPR Technique for Protein Tagging". Cells 9, n.º 12 (5 de diciembre de 2020): 2618. http://dx.doi.org/10.3390/cells9122618.
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Genetic knock-in using homology-directed repair is an inefficient process, requiring the selection of few modified cells and hindering its application to primary cells. Here, we describe Homology independent gene Tagging (HiTag), a method to tag a protein of interest by CRISPR in up to 66% of transfected cells with one single electroporation. The technique has proven effective in various cell types and can be used to knock in a fluorescent protein for live cell imaging, to modify the cellular location of a target protein and to monitor the levels of a protein of interest by a luciferase assay in primary cells.
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Daftarian, Pirouz, Raquibul Chowdhury, Philip Ames, Changli Wei, Alan D. King, Juan Pablo de Rivero Vaccari, Lloye Dillon et al. "In vivo Electroporation and Non-protein Based Screening Assays to Identify Antibodies Against Native Protein Conformations". Hybridoma 30, n.º 5 (octubre de 2011): 409–18. http://dx.doi.org/10.1089/hyb.2010.0120.
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