Los autores desean agradecer a Brian Muntean, Lo Shao, Maki Takahashi y Mell Blair por su asistencia técnica. Este trabajo fue apoyado por concesiones del NIH (DK080640) y la Universidad de Toledo y ProMedica traslacional Premio Estímulo de Investigación con el Dr. Surya Nauli.

1. De la célula pre-tratamiento y preparación de la metafase <ol><li> Las células se cultivan en modif Dulbecco de Medio Eagle (DMEM) que contiene 10-15% de suero fetal bovino (SFB) y 1% penicilina / estreptomicina a 37 ° C con 5% de CO 2 incubadora, hasta que alcanzan 70-80% de confluencia. </li><li> Se tratan las células con solución colcemid a 0,05 ug / ml para 30-60 min. </li><li> Se recoge el medio que contiene las células flotantes en 50 ml tubos de centrífuga estériles de halcón. Lavar las células en el plato con sterile1X-PBS. Después de incubar con tripsina estéril para 1-2 min, se cosecha y recoger las células restantes en el mismo tubo. </li><li> Haga girar los tubos a 1000 rpm durante 5 minutos, aspirar el sobrenadante dejando 0,5 ml y afloje de pellets con un movimiento rápido con el dedo solamente. </li><li> Dependiendo del tamaño de pellets, añadir 5-10 ml de solución hipotónica de 0,56% de KCl en dH 2 O y incubar la suspensión a 37 ° C durante 30-45 min. </li><li> Agregue una gota de metanol / ácido acético(3:1 vol / vol) por ml de suspensión celular hipotónica e invierta el tubo suavemente para mezclar. </li><li> Centrifugar a 1200 rpm durante 5 minutos y recoger el pellet como paso 1.4, a continuación, añadir 5 ml de metanol fresco / ácido acético (3:1 vol / vol) gota a gota la solución de fijación mientras se ojean la pastilla continuamente. Este procedimiento es fundamental para que los spreads metafase no será atrapado en macizos de las células que pondrían en peligro el experimento. </li><li> Centrifugar de nuevo a 1200 rpm durante 5 min y añadir 5-10 ml de helado de metanol / fijador ácido acético a lo largo de la pared del tubo. En esta etapa, si es necesario, las células pueden ser almacenadas en solución fijadora en un tubo apretados y selladas a -20 ° C durante corto plazo o -80 ° C durante largo plazo (años) para su uso futuro. </li><li> Diapositivas Limpieza en etanol absoluto, y luego sumerja la diapositiva en dH 2 O para approximately10X el fin de formar una vaina de agua en la superficie de la diapositiva. Colocar el portaobjetos en una placa de vidrio y la caída del 15-20ul de la suspensión celular (del paso 1.8) De 10 &quot;por encima de la diapositiva. Colocar el portaobjetos en un baño de agua a 65-70 ° C durante 1-2 minutos y dejar secar. </li><li> Compruebe el portaobjetos bajo un microscopio óptico con objetivos secos de 10X y 40X asegurándose de que no hay metafase los cromosomas y los diferenciales de los se espacian uniformemente. Comprobación de la existencia citoplasma que rodea a los cromosomas. Si citoplasma está presente proceder con el pretratamiento de diapositivas (digestión con pepsina), si no hay citoplasma está presente y los cromosomas tienen buena morfología, entonces no hay necesidad de pretratamiento de diapositivas. </li></ol> 2. Deslice el tratamiento previo (pepsina tratamiento) <ol><li> Aplicar 120 l de solución 1:200 RNasa (20 mg / ml) disuelto en 2X-SSC en un 24 mm x 60 mm cubreobjetos microscopio e invierta la cara diapositiva metafase abajo en el cubre objetos a continuación, invierta suavemente la cara diapositiva metafase hasta e incubar a 37 ° C durante 45 min. </li><li> Retire con cuidado el cubreobjetos sin rayar el portaobjetos y lávelo en 2X SSC-tampón en un vaso de Coplin cada 5 minutos durante 15 minutoscon agitación. </li><li> Añadir 5-15 l de solución stock de pepsina (100 mg / ml en dH 2 O) en un vaso de precipitados limpio y luego agregar 100 ml de precalentado (37 ° C) 0,01 M de HCl. Es importante que la pepsina se añade en una limpia vaso de precipitados de primera y no directamente en la solución de HCl, de lo contrario no sería disolver en solución. Incubar el portaobjetos en un vaso de Coplin que contiene la solución de HCl / pepsina a 37 ° C durante 3-5 min. Este paso es muy crítico como la digestión demasiado causará que los cromosomas que se overdigested y demasiado digestión poco va a dejar el citoplasma sin digerir que podría conducir a la unión no específica de la sonda e interferir con la señal de hibridación. </li><li> Lavar el portaobjetos en un vaso de Coplin que contiene 100 ml de 1X-PBS durante 5 minutos dos veces a temperatura ambiente. </li><li> Lavar el portaobjetos en un vaso de Coplin que contiene 100 ml 1X-PBS/MgCl 2 durante 5 min a temperatura ambiente (50 ml de 1M MgCl 2 en 950 ml de 1X-PBS). </li><li> Colocar el portaobjetos en una jarra de Coplin containing 100 ml de 1% de formaldehído durante 10 min a temperatura ambiente (1,7 ml de 37% de formaldehído en 100 ml de 1X-PBS/MgCl 2). </li><li> Lavar el portaobjetos en un vaso de Coplin que contiene 100 ml de 1X-PBS durante 5 min. </li><li> Observe el portaobjetos bajo un microscopio óptico con lente de 40X en seco para asegurar que las diapositivas se digieren correctamente y sin citoplasma está presente y la morfología de los cromosomas se conserva. Seleccione un área para la hibridación con una pluma de diamantes. </li></ol> 3. Cromosoma y la sonda desnaturalización y la hibridación <ol><li> Prepare una solución fresca de desnaturalización (70% formamide/2X SSC, pH 7,0) y precalentamiento a 70-80 ° C en un vaso de Coplin colocado en un Lugar baño de agua la diapositiva en el vaso de Coplin que contiene la solución desnaturalizante en un baño de agua a 70 ° C durante cromosomas de ratón y 80 ° C durante cromosomas humanos para 30s-1.5 min. </li><li> Coloque inmediatamente la diapositiva en el hielo-frío etanol 70% durante 3 min seguido por 80% y 100% de etanol durante 3 min cada uno y aire seco. Examinar el portaobjetospara la morfología cromosoma. Morfología de los cromosomas Bueno se denota por los cromosomas oscuros y no &quot;de luz de fase&quot; o halo cromosomas. </li><li> Caliente la sonda SkyPaint (kit de SKY pintura; vial de # 1) a 37 ° C con agitación durante 20 min, vórtice y centrifugar brevemente a 1000 rpm durante unos pocos segundos. </li><li> Desnaturalizar la sonda en un termociclador programado para un ciclo de dos-paso a 85 ° C para el ciclo de 5 min seguido de 37 ° C durante 60 min para permitir etiquetados-sonda de ADN para preannealing. </li><li> Aplicar 10ul de la sonda desnaturalizada en el área de la hibridación y la tapa con una de 22 mm x 40 mm cubreobjetos con cuidado de no atrapar burbujas de aire. Selle los bordes del cubreobjetos con cemento de goma y se incuban en una cámara húmeda a 37 ° C durante 48-72 horas. </li></ol> 4. La detección de sondas fluorescentes <ol><li> Retire el cubreobjetos con cuidado y coloque el portaobjetos en un vaso de Coplin que contiene precalentado (45 ° C) la solución de lavado I (formamida 50% recién preparada en 2X SSC). Lavar durante 5 minutosrealizada tres veces a 45 ° C en un baño de agua con agitación a 45 rpm </li><li> Lavar el portaobjetos en solución de lavado II (1X SSC) a 45 ° C durante 5 min dos veces con agitación. </li><li> Lave la diapositiva en el lavado de solución III (4X SSC/0.1% de Tween 20) durante 5 min a 45 ° C con agitación. </li><li> Aplique 80 l de reactivo de bloqueo (kit de pintura SKY; vial n º 2), coloque un cubreobjetos y se incuba a 37 ° C durante 30 min. </li><li> Retire la corredera y permitir que el líquido drene. Aplique 80 l de reactivo de tinción Cy5 (concentrado de anticuerpos de detección de CAD kit, vial n º 3), aplique un cubreobjetos y se incuba a 37 ° C durante 40 min. </li><li> Lavar el portaobjetos con solución de lavado III en 45 ° C durante 5 min tres veces con agitación. </li><li> Aplique 80 l de reactivo de tinción Cy5.5 (concentrado de anticuerpos de detección de CAD kit, vial n º 4), coloque un cubreobjetos y se incuba a 37 ° C durante 40 min. </li><li> Lavar el portaobjetos con solución de lavado III en 45 ° C durante 5 min tres veces con agitación. </li><li> Incline la diapositiva y permitir que el líquido<html> escurrir. Aplique 20 l del reactivo anti-fade DAPI (kit de pintura SKY; vial n º 5) y colocar una de 24 mm x 60 mm cubreobjetos microscopio. Retire con cuidado las burbujas de aire que puedan haberse formado. Diapositivas pueden ser reflejados inmediatamente o almacenadas a 4 ° C en la oscuridad durante no más de 1 semana. </li></ol> 5. Imagen de adquisición y análisis <ol><li> La adquisición de imágenes se logra mediante la visualización diapositivas metafase utilizando un microscopio Olympus equipado con una lente de aceite de inmersión 60X, un cubo espectral (personalizado diseñado triples banda filtro de paso), un filtro de DAPI y un módulo de Sagnac interferómetro con una cámara CCD. </li><li> Espectral de los cariotipos se llevaron a cabo utilizando el software SKY Vista (versión aplicada de imágenes espectrales 1,62) siguiendo el manual del usuario. </li><li> Después de analizar las imágenes, los cromosomas pueden ser vistos como imágenes en color (con determinados colores fluorescentes), imágenes de pseudo color (con los colores para la clasificación) y las imágenes invertidas DAPI (patrón específico de bandas). </li></ol>e_title &quot;&gt; 6. Los resultados representativos Un procedimiento de SKY completa generalmente toma alrededor de una vez por semana (Figura 1). Esto incluye la adquisición de imágenes y el análisis siempre que las células en metafase están en cantidad adecuada. Análisis de cariotipo revela cariotipo normal de ratón (40, XY) de las células de los ratones de tipo salvaje (Figura 2a). En contraste, las células de PKD1 - / - ratón (modelo DCP ratón) muestra un aumento significativo en el número de cromosomas y las anormalidades estructurales, tales como deleciones del cromosoma (el cromosoma # 8) y translocaciones (los cromosomas 11 y 19) (Figura 2b). También se analizaron los tejidos vasculares de los pacientes PQRAD. Un estudio simple contando los números cromosómicos indicó que no PQRAD y algunas muestras vasculares PQRAD tenía normales números cromosómicos de 23 pares (figura 2c). En general, sin embargo, se observó fracaso de la segregación cromosómica, lo que resulta en 46 pares de cromosomas en AMuestras DPKD en lugar de pares 23 (Figura 2d). <img alt="Figura 1" src="/files/ftp_upload/3887/3887fig1.jpg" /> Figura 1. Diagrama de flujo de SKY protocolo. Diagrama de flujo del protocolo de SKY ilustra los pasos para completar un experimento a partir de células pre-tratamiento y preparaciones en metafase a la adquisición y análisis de imágenes. Un aproximado de una semana calendario se presenta a la izquierda con los procedimientos paso a paso para cada día. <img alt="Figura 2" src="/files/ftp_upload/3887/3887fig2.jpg" /> Figura 2. Cariotipo espectral en inmortalizado líneas celulares de ratón y células primarias recién aisladas de humanos. El color y las imágenes invertidas DAPI de los cromosomas individuales se muestran antes de la clasificación de los cromosomas. Después de la clasificación, los cromosomas se presentan en la &quot;clasificación&quot; de mesa. Una imagen del cielo) de una extensión metafase de ratón de tipo salvaje contiene un cariotipo normal (40, XY). B) </strong&gt; SKY imagen de una propagación de la metafase PKD1 - / -. del ratón muestra el número de cromosomas anormales (68 en lugar de 40) y las anomalías cromosómicas, como deleciones del cromosoma (el cromosoma 8) y translocaciones cromosómicas (cromosomas 11 y 19) c) SKY imagen de una propagación metafase del tejido vascular de un paciente no tiene PQRAD cariotipo normal (46, XX). d) imagen del cielo de una propagación de la metafase el tejido vascular de un paciente con PQRAD contiene cariotipo anormal (92, XXXX). Partes de los datos han sido previamente informado y se reutiliza con permiso 14.

<ol>
	<li>Caspersson, T., Farber, S., Foley, G. E., Kudynowski, J., Modest, E. J., Simonsson, E., Wagh, U., Zech, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+differentiation+along+metaphase+chromosomes.">Chemical differentiation along metaphase chromosomes.</a> Experimental Cell Research. 49, 219-222 (1968).</li><li>Seabright, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+banding+technique+for+human+chromosomes.">A rapid banding technique for human chromosomes.</a> Lancet. 2, 971-972 (1971).</li><li>Padilla-Nash, H. M., Barenboim-Stapleton, L., Difilippantonio, M. J., Ried, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+karyotyping+analysis+of+human+and+mouse+chromosomes.">Spectral karyotyping analysis of human and mouse chromosomes.</a> Nature Protocols. 1, 3129-3142 (2006).</li><li>Schrock, E., Zschieschang, P., O'Brien, P., Helmrich, A., Hardt, T., Matthaei, A., Stout-Weider, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+karyotyping+of+human,+mouse,+rat+and+ape+chromosomes--applications+for+genetic+diagnostics+and+research.">Spectral karyotyping of human, mouse, rat and ape chromosomes--applications for genetic diagnostics and research.</a> Cytogenetic and Genome Research. 114, 199-221 (2006).</li><li>Padilla-Nash, H. M., Heselmeyer-Haddad, K., Wangsa, D., Zhang, H., Ghadimi, B. M., Macville, M., Augustus, M., Schrock, E., Hilgenfeld, E., Ried, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jumping+translocations+are+common+in+solid+tumor+cell+lines+and+result+in+recurrent+fusions+of+whole+chromosome+arms.">Jumping translocations are common in solid tumor cell lines and result in recurrent fusions of whole chromosome arms.</a> Genes, Chromosomes &#38; Cancer. 30, 349-363 (2001).</li><li>Veldman, T., Vignon, C., Schrock, E., Rowley, J. D., Ried, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hidden+chromosome+abnormalities+in+haematological+malignancies+detected+by+multicolour+spectral+karyotyping.">Hidden chromosome abnormalities in haematological malignancies detected by multicolour spectral karyotyping.</a> Nature Genetics. 15, 406-410 (1997).</li><li>Karpf, A. R., Matsui, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+disruption+of+cytosine+DNA+methyltransferase+enzymes+induces+chromosomal+instability+in+human+cancer+cells.">Genetic disruption of cytosine DNA methyltransferase enzymes induces chromosomal instability in human cancer cells.</a> Cancer Research. 65, 8635-8639 (2005).</li><li>Matsui, S., Faitar, S. L., Rossi, M. R., Cowell, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+spectral+karyotyping+to+the+analysis+of+the+human+chromosome+complement+of+interspecies+somatic+cell+hybrids.">Application of spectral karyotyping to the analysis of the human chromosome complement of interspecies somatic cell hybrids.</a> Cancer Genetics and Cytogenetics. 142, 30-35 (2003).</li><li>Nestor, A. L., Hollopeter, S. L., Matsui, S. I., Allison, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+genetic+complementation+controlling+the+chromosomal+abnormalities+and+loss+of+heterozygosity+formation+in+cancer.">A model for genetic complementation controlling the chromosomal abnormalities and loss of heterozygosity formation in cancer.</a> Cytogenetic and Genome Research. 116, 235-247 (2007).</li><li>Ried, T., Liyanage, M., du Manoir, S., Heselmeyer, K., Auer, K., Macville, M., Schrock, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cytogenetics+revisited:+comparative+genomic+hybridization+and+spectral+karyotyping.">Tumor cytogenetics revisited: comparative genomic hybridization and spectral karyotyping.</a> Journal of Molecular Medicine (Berlin, Germany). 75, 801-814 (1997).</li><li>AbouAlaiwi, W. A., Takahashi, M., Mell, B. R., Jones, T. J., Ratnam, S., Kolb, R. J., Nauli, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliary+polycystin-2+is+a+mechanosensitive+calcium+channel+involved+in+nitric+oxide+signaling+cascades.">Ciliary polycystin-2 is a mechanosensitive calcium channel involved in nitric oxide signaling cascades.</a> Circulation Research. 104, 860-869 (2009).</li><li>Nauli, S. M., Alenghat, F. J., Luo, Y., Williams, E., Vassilev, P., Li, X., Elia, A. E., Lu, W., Brown, E. M., Quinn, S. J., Ingber, D. E., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycystins+1+and+2+mediate+mechanosensation+in+the+primary+cilium+of+kidney+cells.">Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells.</a> Nature Genetics. 33, 129-137 (2003).</li><li>Nauli, S. M., Kawanabe, Y., Kaminski, J. J., Pearce, W. J., Ingber, D. E., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cilia+are+fluid+shear+sensors+that+regulate+calcium+signaling+and+nitric+oxide+production+through+polycystin-1.">Endothelial cilia are fluid shear sensors that regulate calcium signaling and nitric oxide production through polycystin-1.</a> Circulation. 117, 1161-1171 (2008).</li><li>AbouAlaiwi, W. A., Ratnam, S., Booth, R. L., Shah, J. V., Nauli, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+from+humans+and+mice+with+polycystic+kidney+disease+are+characterized+by+polyploidy+and+chromosome+segregation+defects+through+survivin+down-regulation.">Endothelial cells from humans and mice with polycystic kidney disease are characterized by polyploidy and chromosome segregation defects through survivin down-regulation.</a> Human Molecular Genetics. 20, 354-367 (2011).</li><li>Padilla-Nash, H. M., Nash, W. G., Padilla, G. M., Roberson, K. M., Robertson, C. N., Macville, M., Schrock, E., Ried, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cytogenetic+analysis+of+the+bladder+carcinoma+cell+line+BK-10+by+spectral+karyotyping.">Molecular cytogenetic analysis of the bladder carcinoma cell line BK-10 by spectral karyotyping.</a> Genes, Chromosomes &#38; Cancer. 25, 53-59 (1999).</li></ol>

No hay conflictos de interés declarado.

Cariotipo espectral (SKY) es una técnica de citogenética utilizada en el estudio de las composiciones genómicas y cromosómicas. Esta técnica se aprovecha de las sondas de pintado cromosómico, y la detección de estas sondas son adquiridos a través de un interferómetro de Sagnac. El proceso de SKY completa generalmente toma alrededor de una semana, e implica varios pasos clave (Figura 1). El SKY utiliza un protocolo estándar, el cual fue descrito por primera vez por Padilla-Nash et al <...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> DMEM </td><td> Cellgro Mediatech Inc. </td><td> 10 a 013 CV </td><td></td></tr><tr><td> De suero fetal bovino (SFB) </td><td> Hyclone </td><td> SH30088-03 </td><td></td></tr><tr><td> Penecillin / estreptomicina </td><td> Thermo Scientific </td><td> SV30010 </td><td></td></tr><tr><td> Colcemid </td><td> Roche Diagnostics </td><td> 10 295 892 001 </td><td> 10 g / ml </td></tr><tr><td> De HCl </td><td> Fisher Scientific </td><td> A144-500 </td><td></td></tr><tr><td> KCl </td><td> Fisher Scientific </td><td> S77375-1 </td><td></td></tr><tr><td> Fosfato solución salina tamponada </td><td> Thermo Scientific </td><td> SH30256-01 </td><td></td></tr> &lt;tr&gt; <td> SKY pintura Sonda (Humana) </td><td> Imágenes Applied Spectral </td><td> SKY000028 </td><td></td></tr><tr><td> SKY pintura Probe Kit (Mouse) </td><td> Imágenes Applied Spectral </td><td> SKY000030 </td><td></td></tr><tr><td> Concentrado kit de detección de anticuerpos </td><td> Imágenes Applied Spectral </td><td> SKY000033 </td><td></td></tr><tr><td> Tripsina </td><td> Thermo Scientific </td><td> SH30236.01 </td><td></td></tr><tr><td> El metanol </td><td> Fisher Scientific </td><td> A433P-4 </td><td></td></tr><tr><td> El ácido acético </td><td> Fisher Scientific </td><td> A38-212 </td><td></td></tr><tr><td> RNasa A </td><td> Roche Diagnostics </td><td> 10 109 169 001 </td><td></td></tr><tr><td> Pepsina </td><td> Sigma-Aldrich </td><td> P6887-5G </td><td></td></tr><tr><td> MgCl 2 </td><td> Fluka </td><td> 63069-500ML </td><td></td></tr><tr><td> 37% El formaldehído </td><td> Mallinckrodt Baker </td><td> 2106-02 </td><td></td></tr><tr><td> SSC 20X </td><td> Promega </td><td> V4261 </td><td></td></tr><tr><td> Formamida </td><td> Fluka </td><td> 47671 </td><td> preparar justo antes de usarse </td></tr><tr><td> Tween-20 </td><td> Fisher Scientific </td><td> BP337-500 </td><td></td></tr><tr><td> Portaobjetos de vidrio </td><td> Fisher Scientific </td><td> 12-549 </td><td></td></tr><tr><td> Microscopio cubierta de vidrio 24x60mm </td><td> VWR Vista Vision </td><td> 16004-312 </td><td></td></tr><tr><td> Adhesivo de goma </td><td> Elmer (tienda local) </td><td></td><td></td></tr><tr><td> Hibridación / humidifiedchamber / bandeja </td><td> Simport </td><td> M920-2 </td><td> poner las toallas de papel mojadas en la parte inferior </td></tr><tr><td> Thermociclista </td><td> Eppendorf </td><td> Epgradient S </td><td></td></tr><tr><td> Plataforma de agitación / Agitador orbital </td><td> Bellco Biotecnología </td><td></td><td></td></tr><tr><td> Baño de agitación / agua </td><td> Precisión Científico </td><td></td><td></td></tr><tr><td> DAPI filtro de cubo </td><td> Chroma Technology </td><td></td><td></td></tr><tr><td> SKY filtro de cubo </td><td> Chroma Technology </td><td></td><td></td></tr><tr><td> SpectraCube </td><td> Imágenes Applied Spectral </td><td></td><td></td></tr><tr><td> Celular microscopio invertido la cultura </td><td> Nikon </td><td> Nikon Eclipse TS100 </td><td></td></tr><tr><td> Microscopio de fluorescencia </td><td> Olimpo </td><td> IX70 </td><td> Aceite de 60X </td></tr></tbody></table>

spectral karyotyping to study chromosome abnormalities in humans and mice with polycystic kidney disease

El método convencional para identificar y clasificar los cromosomas individuales depende del patrón único de bandas de cada cromosoma en una especie en particular se analizaron 1, 2. Esta técnica clásica anillamiento, sin embargo, no es fiable en la identificación de complejos aberraciones cromosómicas tales como aquellas asociadas con el cáncer. Para superar las limitaciones de la técnica de bandeo, cariotipo espectral (SKY) se introdujo para proporcionar mayor cantidad de información fiable sobre las anomalías cromosómicas. SKY es una fluorescencia multicolor de hibridación in situ (FISH) para detectar los cromosomas en metafase con el microscopio espectral de 3, 4. SKY ha sido demostrado ser una herramienta valiosa para el análisis citogenético de una amplia gama de anomalías cromosómicas asociadas con un gran número de enfermedades genéticas y enfermedades malignas 5, 6. CIELO implica el uso de sondas de ADN multicolores fluorescentemente-etiquetados preparados a partir de la oligonucleótidos degeneradoscebadores otide por PCR. Así, cada cromosoma tiene un color único espectral después de hibridación in situ con sondas, que están diferencialmente etiquetados con una mezcla de tintes fluorescentes (Rodamina, Texas Red, Cy5, FITC y Cy5.5). Las sondas utilizadas para SKY constar de hasta 55 sondas cromosómicas específicas 7-10. El procedimiento para SKY implica varios pasos (Figura 1). CIELO requiere la disponibilidad de células con alto índice mitótico a partir de tejido normal o enferma o sangre. Los cromosomas de una sola célula a partir de ya sea un celular recién aisladas primaria o una línea celular se extienden sobre un portaobjetos de vidrio. Esta propagación cromosoma está etiquetado con una combinación diferente de tintes fluorescentes específicas para cada cromosoma. Para la detección de la sonda y la adquisición de imágenes, el sistema de imagen espectral consta de interferómetro de Sagnac y una cámara CCD. Esto permite la medición del espectro de luz visible emitida desde la muestra y para adquirir una imagen espectral vaivénm individuales cromosomas. HiSKY, el software utilizado para analizar los resultados de las imágenes capturadas, proporciona una fácil identificación de anomalías cromosómicas. El resultado final es una metafase y una imagen de clasificación cariotipo, en el que cada par de cromosomas tiene un color distinto (Figura 2). Esto permite una fácil identificación de las identidades cromosoma y translocaciones. Para más información, visite el sitio web Aplicada Spectral Imaging ( <a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a> ). SKY se utilizó recientemente para la identificación de los defectos de la segregación cromosómica y las anomalías cromosómicas en humanos y ratones con autosómica dominante renal poliquística (PQRAD), una enfermedad genética que se caracteriza por una disfunción en los cilios primarios 11-13. Usando esta técnica, hemos demostrado la presencia de la segregación cromosómica anormal y anomalías cromosómicas en pacientes PQRAD y modelos de ratón 14. Los análisis adicionales utilizando SKY no sólo nos permitió identificar el número cromosómico y la identidad, sino también para detectar con precisión las aberraciones cromosómicas muy complejas, tales como deleciones y translocaciones cromosómicas (Figura 2).

Watch this Scientific Journal Video about Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease at JoVE.com

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Cariotipo espectral (SKY) es una técnica de citogenética avanzada para identificar aberraciones genómicas y cromosómicas. Esta técnica se aprovecha de las sondas de pintado cromosómico, lo que permite la clasificación de todos los cromosomas. Sky también puede identificar aberraciones cromosómicas y defectos complejos de segregación en ratones y seres humanos con diversas enfermedades, incluyendo enfermedad renal poliquística.

Cariotipo espectral para el Estudio de anomalías cromosómicas en humanos y ratones con la enfermedad renal poliquística

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

spectral-karyotyping-to-study-chromosome-abnormalities-humans-mice

Research

JoVE Journal

Medicine

38.2K vistas.  University of Toledo, College of Pharmacy and Pharmaceutical Sciences. Cariotipo espectral (SKY) es una técnica de citogenética avanzada para identificar aberraciones genómicas y cromosómicas. Esta técnica se aprovecha de las sondas de pintado cromosómico, lo que permite la clasificación de todos los cromosomas. Sky también puede identificar aberraciones cromosómicas y defectos complejos de segregación en ratones y seres humanos con diversas enfermedades, incluyendo enfermedad renal poliquística.

Video: Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

El trasplante ortotópico aórtica en los ratones para el Estudio de la Enfermedad Vascular

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

Investigación

Medicina

5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. To study development of CKD and novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well known experimental model of progressive renal disease, resembling several aspects of human CKD. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is also associated with progressive intrarenal capillary loss, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function. To mimic this, we combine removal of 5/6th of renal mass with nitric oxide (NO) depletion and a high salt diet. After arrival and acclimatization, animals receive a NO synthase inhibitor (NG-nitro-L-Arginine) (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy. One week later, a subtotal nephrectomy (SNX) is performed on the left side. After SNX, animals are allowed to recover for two days followed by LNNA in drinking water (20 mg/L) for a further period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed. Renal function is measured using 'gold standard' inulin and para-amino hippuric acid (PAH) clearance technology. This model of CKD is characterized by a reduction in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), hypertension (systolic blood pressure&gt;150 mmHg), proteinuria (&gt; 50 mg/24 hr) and mild uremia (&gt;10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (&lt;10%). Follow-up until 12 weeks after SNX shows further progression of CKD.

A two-stage method to establish chronic kidney disease (CKD) in the Lewis rat by surgically removing 5/6th of renal mass is described. Combination of the surgical procedure, NOS-inhibition and a high-salt diet leads to a model resembling human CKD, allowing study of causal mechanisms and development of novel therapeutic interventions.

5/6th nefrectomía en combinación con dieta alta en sal y el óxido nítrico sintasa inhibición para inducir la enfermedad renal crónica en la rata Lewis

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

Evaluación de la función vascular en pacientes con enfermedad renal crónica

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

Un posible modelo de pez cebra de la enfermedad renal poliquística: Derribo de<em&gt; Wnt5a</em&gt; Causas Los quistes en el pez cebra Riñones

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

El uso de ultra-alta MRI Field en Modelos pequeño roedor de la enfermedad renal poliquística para<em&gt; En Vivo</em&gt; Fenotipificación de Drogas y Vigilancia

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Symmetric Bihemispheric Postmortem Brain Cutting to Study Healthy and Pathological Brain Conditions in Humans

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena described in those patients for whom a brain autopsy has been requested. Although the advancements of biomedical sciences and neuroimaging have revolutionized the neuropsychiatric field, they have also generated the misleading idea that brain autopsies have only a confirmatory value. This false idea created a drastic reduction of autopsy rates and, consequently, a reduced possibility to perform more detailed and extensive neuropathological investigations, which are necessary to comprehend numerous normal and pathological aspects yet unknown of the human brain. The traditional inferential method of correlation between observed neuropsychiatric phenomena and corresponding localization/characterization of their possible neurohistological correlates continues to have an undeniable value. In the context of neuropsychiatric diseases, the traditional clinicopathological method is still the best possible methodology (and often the only available) to link unique neuropsychiatric features to their corresponding neuropathological substrates, since it relies specifically upon the direct physical assessment of brain tissues. The assessment of postmortem brains is based on brain cutting procedures that vary across different neuropathology centers. Brain cuttings are performed in a relatively extensive and systematic way based on the various clinical and academic contingencies present in each institution. A more anatomically inclusive and symmetric bi-hemispheric brain cutting methodology should at least be used for research purposes in human neuropathology to coherently investigate, in depth, normal and pathological conditions with the peculiarities of the human brain (i.e., hemispheric specialization and lateralization for specific functions). Such a method would provide a more comprehensive collection of neuropathologically well-characterized brains available for current and future biotechnological and neuroimaging techniques. We describe a symmetric bi-hemispheric brain cutting procedure for the investigation of hemispheric differences in human brain pathologies and for use with current as well as future biomolecular/neuroimaging techniques.

Organized brain cutting procedures are necessary to correlate specific neuropsychiatric phenomena with definitive neuropathologic diagnoses. Brain cuttings are performed differently based on various clinico-academic contingencies. This protocol describes a symmetric bihemispheric brain cutting procedure to investigate hemispheric differences in human brain pathologies and to maximize current and future biomolecular/neuroimaging techniques.

Simétrica bihemisférica post-mortem del cerebro de corte para estudiar sana y patológica condiciones cerebrales en humanos

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena ...

A Model to Simulate Clinically Relevant Hypoxia in Humans

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to hypoxia sensitive organs such as the brain, compensatory circulatory mechanisms help to maintain an adequate oxygen supply. This is mainly achieved by increased cerebral blood flow. Intermittent hypoxia is a commonly seen phenomenon in patients with obstructive sleep apnea. Acute airway obstruction can also result in hypoxia and hypercapnia. Until now, no adequate model has been established to simulate these dynamics in humans. Previous investigations focusing on human hypoxia used inhaled hypoxic gas mixtures. However, the resulting hypoxia was combined with hyperventilation and is therefore more representative of high altitude environments than of apnea. Furthermore, the transferability of previously performed animal experiments to humans is limited and the pathophysiological background of apnea induced physiological changes is poorly understood. In this study, healthy human apneic divers were utilized to mimic clinically relevant hypoxia and hypercapnia during apnea. Additionally, pulse-oximetry and Near Infrared Spectroscopy (NIRS) were used to evaluate changes in cerebral and peripheral oxygen saturation before, during, and after apnea.

Hypoxia simulation in humans has usually been performed by inhaling hypoxic gas mixtures. For this study, apneic divers were used to simulate dynamic hypoxia in humans. Additionally, physiological changes in desaturation and re-saturation kinetics were evaluated with non-invasive tools such as Near-Infrared-Spectroscopy (NIRS) and peripheral oxygenation saturation (SpO2).

Un modelo para simular la hipoxia clínicamente relevantes en seres humanos

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

En Vivo Proyección de imagen de ratones de reportero Cx3cr1gfp/gfp con tomografía de coherencia óptica de dominio espectral y exploración oftalmoscopia de láser

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

Evaluación de la función renal en modelos de ratón de la enfermedad Glomerular

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Delivery of Exogenous Artificially Synthesized miRNA Mimic to the Kidney Using Polyethylenimine Nanoparticles in Several Kidney Disease Mouse Models

microRNAs&#160;(miRNAs), small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by destabilizing and inhibiting their translation in various kidney diseases. Therefore, alternation of miRNA expression by exogenous artificially synthesized miRNA mimics is a potentially useful treatment option for inhibiting the development of many kidney diseases. However, because serum RNAase immediately degrades systematically administered exogenous miRNA mimics in vivo, delivery of miRNA to the kidney remains a challenge. Therefore, vectors that can protect exogenous miRNA mimics from degradation by RNAase and significantly deliver them to the kidney are necessary. Many studies have used viral vectors to deliver exogenous miRNA mimics or inhibitors to the kidney. However, viral vectors may cause an interferon response and/or genetic instability. Therefore, the development of viral vectors is also a hurdle for the clinical use of exogenous miRNA mimics or inhibitors. To overcome these concerns regarding viral vectors, we developed a nonviral vector method to deliver miRNA mimics to the kidney using tail vein injection of polyethylenimine nanoparticles (PEI-NPs), which led to significant overexpression of target miRNAs in several mouse models of kidney disease.

Here, we deliver exogenous artificially synthesized miRNA mimics to the kidney via tail vein injection of a nonviral vector and polyethylenimine nanoparticles in several kidney disease mouse models. This led to significant overexpression of target miRNA in the kidney, resulting in inhibited progression of kidney disease in several mouse models.

Entrega de miRNA sintetizado artificialmente exógeno imitador al riñón utilizando nanopartículas de polietilenimina en varios modelos de ratón con enfermedad renal

microRNAs&#160;(miRNAs), small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by ...