Este trabajo fue apoyado por la Agencia de Investigación de Eslovenia (proyecto J2-9770, IP-0510 centro de la infraestructura y el programa P2-0249). Este video representa el material complementario para la &quot;base de electroporación, tecnologías y tratamientos&quot; taller científico y postgrado, organizado por la Facultad de Ingeniería Eléctrica de la Universidad de Ljubljana, en Eslovenia. Los autores agradecen también Dusa Hodzic por la amabilidad de proporcionar el ADN del plásmido.

1. Cultivo de células, plásmido y la preparación de amortiguamiento para el experimento <ol><li> En este experimento células ováricas de hámster chino (CHO-K1) se utilizan. Las células se cultivan en una mezcla de nutrientes HAM-F12 (PAA) suplementado con 2 mM L-glutamina, un 10% de suero fetal bovino, 400 l / l de gentamicina (todos de Sigma-Aldrich Chemie GmbH, Deisenhofen, Alemania), y 1 ml / l crystacilin (Pliva, Zagreb, Croacia). Las células se mantuvieron a 37 º C en un humidificado 5% de CO2 la atmósfera en la incubadora durante 24 horas. </li><li> Amplificar plásmido pEGFP-N1 (Clontech Laboratories Inc., Mountain View, CA, EE.UU.) que codifica la proteína verde fluorescente (GFP) en la cepa de Escherichia coli DH5a y aislarlo con Kit Hi-Speed ​​Maxi plásmido (Qiagen, Hilden, Alemania). Concentración de ADN plásmido (plásmido disuelto en buffer TE) debe ser determinado por espectrofotometría a 260 nm y se confirmó por electroforesis en gel. </li><li> Prepare el buffer de isoosmolar fosfato de sodio (10 mM Na 2 HPO 4, 10 mM NaH 2 PO 4, 1 mM MgCl 2, 250 mM de sacarosa, pH 7,4). </li><li> En el día del experimento de preparar la suspensión de células por tripsinización con 0,25% de tripsina / EDTA (Sigma-Aldrich Chemie GmbH, Deisenhofen, Alemania). Las células se centrifuga durante 5 min a 1000 rpm (180 xg) a 4 ° C (Sigma, Alemania) y resuspender el sedimento celular en tampón fosfato de sodio isoosmolar a una densidad celular de 5 x 10 6 células / ml. </li></ol> 2. Equipo de hardware <ol><li> Las células son expuestas a campos eléctricos en la punta de la pipeta (Figura 1) con electrodos integrados conectados a un generador prototipo de alta tensión. La punta y la geometría de los electrodos permite la aplicación de campo eléctrico relativamente homogénea y el generador permite el envío de pulsos eléctricos en diferentes direcciones. La punta y el generador se desarrollaron en el Laboratorio de Biocibernética, Facultad de Ingeniería Eléctrica de la Universidad de Ljubljana 1. </li></ol><img src="/files/ftp_upload/3309/3309fig1.jpg" alt="Figura 1"/> Figura 1. Vertical y horizontal (a) sección transversal y la fotografía (b) de la punta de la pipeta con electrodos integrados. En la sección transversal de color gris se utiliza para la carcasa de plástico y el negro para los electrodos. La punta de la pipeta con electrodos integrados consta de cuatro electrodos de barra cilíndrica. Los electrodos son de acero inoxidable, su diámetro es de 1,4 mm, electrodos adyacentes son 1 mm, y los electrodos opuestos son 2 mm. Los electrodos se pegan en la punta de plástico en paralelo y su longitud de aplicación es de 30 mm 2. 3. Gene electrotransferencia protocolo <ol><li> Añadir plásmido pEGFP-N1 a una suspensión celular en la concentración de 10 ug / ml. </li><li> Incubar la mezcla durante 2-3 minutos a temperatura ambiente, antes de la aplicación de pulsos eléctricos. </li><li> Aspire 100 l de suspensión de células en la punta de la pipeta con electrodos integrados. </li><li> Para lograr la mejor eficacia de electrotransferencia gen y mantener la viabilidad celular, los parámetros óptimos de los impulsos eléctricos se debe utilizar. En este experimento, un tren de 8 pulsos rectangulares (cada uno con duración de 1 ms, la amplitud de 225 V a una frecuencia de repetición Hz) se aplica a cada muestra, utilizando alta tensión del generador prototipo. Dos diferentes protocolos de campo eléctrico (Figura 2) se utilizan: en el primer protocolo de todos los pulsos son entregados en la misma dirección, mientras que en los pulsos de segundo protocolo se entregan al cambiar alternativamente la dirección del campo eléctrico y la orientación. El segundo protocolo sólo se puede usar con generador de impulsos adecuados, lo que permite la aplicación de pulsos eléctricos en diferentes direcciones. </li><li> Inmediatamente después de la aplicación de pulsos de transferencia de las células de la punta de la pipeta en 6 pocillos y añadir suero fetal bovino (FCS-Sigma, EE.UU.) (25% del volumen de la muestra). </li><li> Se incuban las células durante 5 min a 37 ° C para permitir que el resellado de la membrana celular. </li><li> Añadir 2 ml de HAM-F12 para cada muestra en 6 pocillo e incubar las células durante 24 horas a 37 ° C en un humidificado 5% de CO2 la atmósfera en la incubadora. </li></ol><img src="/files/ftp_upload/3309/3309fig2.jpg" alt="Figura 2"/> . Figura 2 protocolos de campo eléctrico: (a) todos los pulsos son entregados en la misma dirección, (b) Los pulsos son entregados por el cambio, alternativamente, la dirección del campo eléctrico y la orientación. 4. De adquisición de imágenes y la determinación de la eficacia del gen electrotransferencia <ol><li> La eficacia del gen electrotransferencia se determina como el porcentaje de células que expresan GFP 24 horas después de la aplicación de pulsos. </li><li> Las células se observan con un microscopio de fluorescencia (en nuestro caso Zeiss 200, Axiovert, ZR Alemania) con luz de excitación a 488 nm generada por un sistema monocromador (policroma IV, Visitron, Alemania) y la emisión se detecta a 507 nm. Las imágenes se graban mediante imágenes de sistema (sistema de imagen MetaMorph, Visitren, Alemania), pero otro software de adquisición similar también se puede utilizar. </li><li> Adquirir al menos cinco imágenes (contraste de fases y fluorescencia verde) en el aumento del objetivo de 20x. </li><li> Recuento de células en la imagen de contraste de fase y las células que expresan GFP en la imagen de fluorescencia verde. Determinar el porcentaje de eficacia del gen electrotransferencia dividiendo el número de células que expresan GFP con el número de todas las células en cada imagen correspondiente (Figura 3). </li></ol> 5. Los resultados representativos: <img src="/files/ftp_upload/3309/3309fig3.jpg" alt="Figura 3"/> Figura 3. El porcentaje de células que expresan GFP cuando todos los pulsos son entregados en la misma dirección y, cuando las leguminosas son entregados por el cambio, alternativamente, la dirección del campo eléctrico y la orientación se presenta. Las células fueron expuestas a un tren de ocho pulsos con amplitud de 225 V, la duración de 1 ms y la frecuencia de repetición de 1 Hz. Los resultados fueron obtenidos por medio de microscopía de fluorescencia. Cada valor en el gráfico representan la media de tres experimentos independientes ± desviación estándar. Al cambiar la dirección del campo eléctrico y la orientación que el porcentaje de células que expresan GFP aumenta.

<ol>
	<li>Rebersek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporator+with+automatic+change+of+electric+field+direction+improves+gene+electrotransfer+in-vitro.">Electroporator with automatic change of electric field direction improves gene electrotransfer in-vitro.</a> Biomed Eng Online. 6, 25-25 (2007).</li><li>Trontelj, K., Rebersek, M., Miklavcic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tip+electrode+chamber+for+small+volume+electroporation,+electrofusion,+and+gene+transfection.">Tip electrode chamber for small volume electroporation, electrofusion, and gene transfection.</a> 18, (2006).</li><li>Neumann, E., Schaefer-Ridder, M., Wang, Y., Hofschneider, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+mouse+lyoma+cells+by+electroporation+in+high+electric+fields.">Gene transfer into mouse lyoma cells by electroporation in high electric fields.</a> EMBO J. 1, 841-841 (1982).</li><li>Daud, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+I+trial+of+interleukin-12+plasmid+electroporation+in+patients+with+metastatic+melanoma.">Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma.</a> J Clin Oncol. 26, 5896-5903 (2008).</li><li>Pavlin, M., Miklavcic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+conductivity+of+a+suspension+of+permeabilized+cells:+a+theoretical+analysis.">Effective conductivity of a suspension of permeabilized cells: a theoretical analysis.</a> Biophys J. 85, 719-729 (2003).</li><li>Xie, T., Sun, L., Zhao, H., Fuchs, J., Tsong, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+mechanisms+of+electric+field-induced+DNA+transfection.+IV.+Effects+of+DNA+topology+on+cell+uptake+and+transfection+efficiency.">Study of mechanisms of electric field-induced DNA transfection. IV. Effects of DNA topology on cell uptake and transfection efficiency.</a> Biophys J. 63, 1026-1031 (1992).</li><li>Rols, M., Delteil, C., Serin, G., Teissie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+effects+on+electrotransfection+of+mammalian+cells.">Temperature effects on electrotransfection of mammalian cells.</a> Nucleic Acids Res. 22, 540-540 (1994).</li><li>Golzio, M., Teissie, J., Rols, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+synchronization+effect+on+mammalian+cell+permeabilization+and+gene+delivery+by+electric+field.">Cell synchronization effect on mammalian cell permeabilization and gene delivery by electric field.</a> Biochim Biophys Acta. 1563, 23-28 (2002).</li><li>Haberl, S., Miklavcic, D., Pavlin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Mg+ions+on+efficiency+of+gene+electrotransfer+and+on+cell+electropermeabilization.">Effect of Mg ions on efficiency of gene electrotransfer and on cell electropermeabilization.</a> Bioelectrochemistry. 79, 265-271 (2010).</li><li>Rols, M., Teissie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electropermeabilization+of+mammalian+cells+to+macromolecules:+control+by+pulse+duration.">Electropermeabilization of mammalian cells to macromolecules: control by pulse duration.</a> Biophys J. 75, 1415-1423 (1998).</li><li>Kanduser, M., Miklavcic, D., Pavlin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+involved+in+gene+electrotransfer+using+high-and+low-voltage+pulses-An+in+vitro+study.">Mechanisms involved in gene electrotransfer using high-and low-voltage pulses-An in vitro study.</a> Bioelectrochemistry. 74, 265-271 (2009).</li><li>Pavlin, M., Flisar, K., Kanduser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+electrophoresis+in+gene+electrotransfer.">The role of electrophoresis in gene electrotransfer.</a> J Membr Biol. 236, 75-79 (2010).</li><li>Sersa, G., Cemazar, M., Semrov, D., Miklavcic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changing+electrode+orientation+improves+the+efficacy+of+electrochemotherapy+of+solid+tumors+in+mice.">Changing electrode orientation improves the efficacy of electrochemotherapy of solid tumors in mice.</a> Bioelectrochem Bioenerg. 39, 61-66 (1996).</li><li>Faurie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electro-mediated+gene+transfer+and+expression+are+controlled+by+the+life-time+of+DNA/membrane+complex+formation.">Electro-mediated gene transfer and expression are controlled by the life-time of DNA/membrane complex formation.</a> J Gene Med. 12, 117-125 (2010).</li></ol>

No hay conflictos de interés declarado.

Gene electrotransferencia es una técnica versátil que la biotecnología permite la transferencia de ADN en las células mediante la aplicación de pulsos cortos, de alta tensión eléctrica 3 y representa una alternativa más segura a los vectores virales, debido a su seguridad, eficacia y facilidad de aplicación. Aunque hoy en día electrotransferencia gen es ampliamente utilizado para transfectar todos los tipos de células y la primera fase I de ensayos clínicos con este método se ha reportado 4,...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> HAM-F12 </td><td> PAA </td><td> E15-016 </td><td> medio de cultivo </td></tr><tr><td> L-glutamina </td><td> Sigma-Aldrich </td><td> G7513 </td><td></td></tr><tr><td> de suero fetal bovino </td><td> PAA </td><td> A15-151 </td><td></td></tr><tr><td> gentamicina </td><td> Sigma-Aldrich </td><td> G1397 </td><td> antibiótico </td></tr><tr><td> crystacilin </td><td> Pliva </td><td> 625110 </td><td> antibiótico </td></tr><tr><td> pEGFP-N1 </td><td> Clontech Laboratories </td><td> 6085-1 </td><td> ADN plásmido </td></tr><tr><td> Kit Hi-Speed ​​Maxi plásmido </td><td> Qiagen </td><td> 12662 </td><td></td></tr><tr><td> Na 2 HPO 4 </td><td> Merck </td><td> F640786 933 </td><td></td></tr><tr><td> NaH 2 PO 4 </td><td> TKI Hrastnik </td><td> 0795 </td><td></td></tr><tr><td> MgCl2 </td><td> Sigma-Aldrich </td><td> M-8266 </td><td></td></tr><tr><td> sacarosa </td><td> Sigma-Aldrich </td><td> 16104 </td><td></td></tr><tr><td> tripsina / EDTA </td><td> Sigma-Aldrich </td><td> T4174 </td><td></td></tr><tr><td> punta de la pipeta </td><td> Una medida que se </td><td></td><td></td></tr><tr><td> eléctrica del generador de impulsos </td><td> Una medida que se </td><td></td><td></td></tr><tr><td> 6 pocillos </td><td> TPP </td><td> 92406 </td><td></td></tr><tr><td> 15 ml tubo de centrífuga </td><td> TPP </td><td> 91015 </td><td></td></tr><tr><td> 75 cm 2 frasco de la cultura </td><td> TPP </td><td> 90076 </td><td></td></tr></tbody></table>

changing the direction and orientation of electric field during electric pulses application improves plasmid gene transfer in vitro

Gene electrotransferencia es un método físico utilizado para introducir genes en las células mediante la aplicación de pulsos eléctricos cortos e intensos, que provocan la desestabilización de la membrana celular, por lo que es permeable a pequeñas moléculas y permite la transferencia de moléculas grandes como el ADN. Representa una alternativa a los vectores virales, debido a su seguridad, eficacia y facilidad de aplicación. Para el gen de electrotransferencia diferentes protocolos de impulsos eléctricos se utilizan con el fin de lograr la máxima transfección de genes, uno de ellos es cambiar la dirección del campo eléctrico y la orientación durante la entrega de pulso. Cambio de la dirección del campo eléctrico y la orientación de aumentar la superficie de la membrana competente para la entrada de ADN en la célula. En este video, se demuestra la diferencia en la eficacia del gen electrotransferencia cuando todos los pulsos son entregados en la misma dirección y, cuando las leguminosas son entregados por el cambio, alternativamente, la dirección del campo eléctrico y la orientación. Para este propósito la punta con electrodos integrados y de alta tensión del generador prototipo, que permite cambiar de campo eléctrico en diferentes direcciones durante la aplicación de pulsos eléctricos, fueron utilizados. Gene eficacia electrotransferencia se determina 24 horas después de la aplicación de pulsos como el número de células que expresan la proteína verde fluorescente dividido por el número de todas las células. Los resultados muestran que la transfección de genes se incrementa cuando la orientación del campo eléctrico durante la entrega de impulsos eléctricos se cambia.

Watch this Scientific Journal Video about Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro at JoVE.com

Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

Transfección de genes mediante electroporación se mejora aproximadamente dos veces en la orientación del campo eléctrico se modifica durante la aplicación de pulsos, mientras que la viabilidad celular no se ve afectada. El aumento de la transfección de genes es causada por el aumento de la superficie de membrana que se hace competente para la entrada de ADN en la célula.

Cambio de la dirección y la orientación del campo eléctrico durante la aplicación pulsos eléctricos Mejora la transferencia de genes plásmido In vitro</em

Gene electrotransfer is a physical method used to deliver genes into the cells by application of short and intense electric pulses, which cause ...

changing-direction-orientation-electric-field-during-electric-pulses

Research

JoVE Journal

Medicine

Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

10.4K vistas.  University of Ljubljana. Transfección de genes mediante electroporación se mejora aproximadamente dos veces en la orientación del campo eléctrico se modifica durante la aplicación de pulsos, mientras que la viabilidad celular no se ve afectada. El aumento de la transfección de genes es causada por el aumento de la superficie de membrana que se hace competente para la entrada de ADN en la célula.

Video: Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Establecimiento y propagación de los tumores del retinoblastoma humanos en ratones con deficiencia inmune

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Investigación

Medicina

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Transferencia génica para la insuficiencia cardiaca isquémica en un modelo preclínico

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Producción y la detección de especies reactivas del oxígeno (ROS) en los cánceres

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcadores en un modelo animal para Revelar correlatos neurales, hematológicas y conductuales de estrés postraumático

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes.
Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression1. They lack chronic toxicity because they lack viral coding sequences2-5 and retain only Ad cis elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes.
In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice.

We describe hepatic neo-islet formation in STZ (streptozotocin)-induced diabetic mice by gene transfer of Neurogenin3 (Ngn3) and Betacellulin (Btc) using helper-dependent adenoviral vector (HDAd) and the reversal of hyperglycemia. Our method takes advantages of helper-dependent adenoviral vectors with their highly efficient in vivo transduction and the long lasting gene expression.

Neo-Islet Formación en el hígado de ratones diabéticos por Ayudante dependiente de transferencia adenoviral Gene mediada por vector

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the ...

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr&#160;and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.

These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries.

Transferencia génica vascular de Stent metálico superficies utilizando Adenoviral Vectores anclada a través hidrolizables reticulantes

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

A Novel<em&gt; In vivo</em&gt; Técnica de transferencia de genes y<em&gt; In vitro</em&gt; ensayos basados ​​en células para el estudio de la pérdida ósea en los trastornos musculoesqueléticos

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

Increasing Pulmonary Artery Pulsatile Flow Improves Hypoxic Pulmonary Hypertension in Piglets

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular failure. Current treatments are limited. Physiologically, pulsatile blood flow is detrimental to the vasculature. In response to sustained pulsatile stress, vessels release nitric oxide (NO) to induce vasodilation for self-protection. Based on this observation, this study developed a protocol to assess whether an artificial pulmonary pulsatile blood flow could induce an NO-dependent decrease in pulmonary artery pressure. One group of piglets was exposed to chronic hypoxia for 3 weeks and compared to a control group of piglets. Once a week, the piglets underwent echocardiography to assess PAH severity. At the end of hypoxia exposure, the piglets were subjected to a pulsatile protocol using a pulsatile catheter. After being anesthetized and prepared for surgery, the jugular vein of the piglet was isolated and the catheter was introduced through the right atrium, the right ventricle and the pulmonary artery, under radioscopic control. Pulmonary artery pressure (PAP) was measured before (T0), immediately after (T1) and 30 min after (T2) the pulsatile protocol. It was demonstrated that this pulsatile protocol is a safe and efficient method of inducing a significant reduction in mean PAP via an NO-dependent mechanism. These data open up new avenues for the clinical management of PAH.

Pulmonary hypertension is associated with a significant reduction in pulmonary artery pulsatility, contractility and elasticity, contributing to an increase in pulmonary artery pressure and pulmonary resistance. Using a hypoxic piglet model, this study demonstrated that improving pulmonary artery plasticity using a newly developed pulsatile catheter improves hypoxic pulmonary hypertension.

El aumento de la arteria pulmonar de flujo pulsátil Mejora hipóxico hipertensión pulmonar en lechones

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular ...

Adapted Resistance Training Improves Strength in Eight Weeks in Individuals with Multiple Sclerosis

Hip weakness is a common symptom affecting walking ability in people with multiple sclerosis (MS). It is known that resistance strength training (RST) can improve strength in individuals with MS, however; it remains unclear the duration of RST that is needed to make strength gains and how to adapt hip strengthening exercises for individuals of varying strength using only resistance bands. This paper describes the methodology to set up and implement an adapted resistance strength training program, using resistance bands, for individuals with MS. Directions for pre- and post-strength tests to evaluate efficacy of the strength-training program are included. Safety features and detailed instructions outline the weekly program content and progression. Current evidence is presented showing that significant strength gains can be made within 8 weeks of starting a RST program. Evidence is also presented showing that resistance strength training can be successfully adapted for individuals with MS of varying strength with little equipment.

Hip weakness is a common symptom affecting walking ability in people with multiple sclerosis. Isolated muscle strengthening is a useful method to target specific weaknesses. This protocol describes a progressive resistance-training program using exercise bands to increase hip muscle strength.

Entrenamiento de la resistencia Adaptado mejora la fuerza en ocho semanas en personas con esclerosis múltiple

Hip weakness is a common symptom affecting walking ability in people with multiple sclerosis (MS). It is known that resistance strength training (RST) can ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

El agotamiento de las células de ratón a partir de xenoinjertos de tumores humanos mejora significativamente análisis de aguas abajo de las células diana

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...