Este trabajo fue apoyado por el NIH (DK093625 a la LH, y DK069909 y DK047757 a JHL) y la Administración de Veteranos (Premio al Mérito I01BX000820 a JHL). Nos gustaría agradecer al Dr. Michael paquete y el Dr. Jie Él en la Universidad de Pennsylvania pez cebra Core para proporcionar un apoyo esencial.

Declaración de Ética: NOTA: Todos los experimentos de pez cebra fueron aprobados por el Comité de Cuidado y Uso de Animales institucional en la Eastern Virginia Medical School. 1. Preparación morfolino <ol><li> Diseño y síntesis de traducción-bloqueo (aumentación) y el empalme de inhibición (Splice-) morfolino anti-sentido (MO) oligonucleótidos para el gen de interés según las instrucciones del fabricante (Figura 1A). Por favor, consulte la información del fabricante en la Tabla 1. NOTA: OM se envían como acciones liofilizados en botellas de vidrio. </li><li> Añadir alto grado agua estéril a las botellas de vidrio para volver a suspender MOS a una concentración final de 25 mg / l. Asegúrese de que el oligonucleótido se disuelva por completo. Si algunos restos sólidos, caliente el frasco que contiene la solución de oligonucleótido a 65 ° C durante 5 a 10 minutos y agitar brevemente. </li><li> La solución madre, MO en RT. No se puede almacenar a 4 ° C o -20 °; C porque las temperaturas más bajas podrían causar el oligonucleótido MO para unirse a la pared del recipiente. Medir la concentración de la solución madre usando un espectrofotómetro (véase Tabla 1) cada vez que se realiza una nueva MO solución madre. </li><li> Preparar la solución de trabajo de MO en el día de la inyección mediante la dilución de la solución madre con alto grado de agua estéril a la dosis deseada. Añadir 0,5% de rojo de fenol para alcanzar una concentración de 0,05%. Por ejemplo, para hacer que la solución de trabajo 5 l con una concentración de 15 ng / nL, agregue 3 l MO solución stock y 0,5 l 0,5% de rojo de fenol al 1,5 l de agua. NOTA: Con esta concentración de trabajo, cada gota (500 pl) de inyección contiene 7,5 ng de MO y dos gotas contiene 15 ng de MO. </li></ol> 2. Preparación del aparato de inyección <ol><li> Compra de vidrio agujas pre-tiró (véase el cuadro 1 para información detallada). Como alternativa, retire la aguja de inyección wi vidrioth un extractor de aguja. </li><li> Encienda el compresor de aire y ajustar el valor a 50 psi de presión. Encienda la fuente de luz del microscopio de disección y Pico bomba de microinyección y ajustar la configuración de la siguiente manera: mantener la presión de 20 psi, expulsar la presión de 10 psi, valor del período de 2,5 y 100 microsegundos gama de gating. </li><li> Cargar el vidrio aguja pre-desmenuzado con 5 l de la solución de inyección y colocar la aguja en una posición vertical con la punta hacia abajo. Espere hasta que toda la solución llega a la punta de la aguja sin burbujas de aire visibles. Coloque el soporte de la aguja en una posición apropiada al lado del microscopio e inserte la aguja de vidrio en el soporte. Ajuste el ángulo de inyección a 45 grados. </li><li> Llevar la punta de la aguja a la vista bajo el microscopio, alto del escenario, y se centran en la región más delgada de la punta. Rompa la punta de la aguja con unas pinzas de punta fina. </li><li> Coloque una regla y un tubo capilar con diámetro interior de 0,15 mm de lado a lado con el microscopio;inyectar la solución en un extremo del tubo capilar para hacer una columna de líquido. Ajustar el tiempo de expulsión para llegar a cada 17 gotas por la columna de líquido de longitud 1 mm, entonces el volumen de cada gota es 1 nl (volumen de la gota = π x radio de 2 x longitud (de la columna de líquido) / count (de gotas). NOTA: Una gota con un diámetro de 0,1 mm da un volumen de inyección de alrededor de 500 pl (volumen de la gota = 4/3 x π x radio de 3). </li></ol> 3. Preparación de fertilizados embriones de pez cebra y de inyección con Morpholinos <ol><li> Configure Tg (wt1b: GFP) parejas reproductoras de pez cebra transgénico de acuerdo con los protocolos publicados para la cría de peces cebra estándar y mantenimiento. Recoger los embriones después de desove natural, y mantenerlos en una placa de Petri de 10 cm llena de agua E3 (5 mM NaCl, KCl 0,17 mm, 0,33 mM CaCl 2, 0,33 mM MgSO4). Iniciar MO inyección de inmediato 13. Monitorear morfología del embrión bajo el microscopio y hacer la inyección segura de MO es en el de unoetapa de célula, o no más tarde de la etapa de cuatro células. <ol><li> Transferir los embriones a una placa de Petri de 10 cm con forma de cuña agar valles. Aspirar el agua E3 extra y presionar suavemente los embriones en los comederos. </li><li> Manipular los embriones con la micropipeta para visualizar embriones en estadio de 1 célula bajo el microscopio de disección. Penetrar en el corion y luego la yema para inyectar una o dos gotas de MO en la yema (1 gota = 500 pl). La transferencia de los embriones inyectados a un 10 cm placa de Petri con agua E3 y se incuba a 28,5 ° C. </li></ol></li><li> Después de las inyecciones, toma de embriones muertos y registrar el número de embriones inyectados. Cambie el agua E3 en el plato de cada 24 a 48 horas. </li></ol> 4. Experimentos fenotipo de rescate <ol><li> Seleccione un orthologue de otra especie que tiene una estructura de base par primario diferente (generalmente 3-7 pares de bases desajustes) y es, por lo tanto, resistentes a la MO, para el rescate. Por ejemplo, en este experimento, elegimosratón porque la secuencia de ratón Wnt5a ARNm es diferente de la secuencia de pez cebra. </li><li> Diseñar un conjunto de cebadores con el cebador directo comenzando en el sitio de la traducción y el cebador inverso en cerca del final de la región codificante del mRNA. Añadir una secuencia de promotor T7 en el extremo 5 &#39;final de la secuencia del cebador hacia adelante. En este experimento, se utilizaron los siguientes el primer secuencias; adelante: 5&#39;-taa tac gac tca etiqueta cta GGA cta tga tgc tgc tga agc tga a- 3 &#39;y revertir: 5&#39;-tca ctt gca gac gta CTG gtc- 3&#39;. </li><li> Realizar una reacción en cadena de polimerasa 50 l (PCR) con el conjunto de cebadores (0.5 M de cada cebador) diseñado arriba y 0.1 g de ADN plantilla que contiene la secuencia de ADNc de ratón Wnt5a con el siguiente programa de PCR: 1 ciclo a 95 ° C durante 5 min; 35 ciclos de 95 ° C (30 seg), 58,5 ° C (30 segundos), 72 ° C (1 min); y paso de extensión final a 72 ° C durante 7 minutos. </li><li> Después de terminar tél ciclo, ejecute 2 l de producto de PCR en un 1% en gel de agarosa para asegurarse de que la banda es el tamaño correcto. Se purifica el producto de PCR con un kit de purificación de PCR de acuerdo con las instrucciones del fabricante. Comprueba la concentración de ADN con un espectrofotómetro. Almacenar el producto de PCR purificado a -20 ° C o usar directamente en la transcripción de ARN tapado en la siguiente etapa. </li><li> Sintetizar in vitro capsulado ARNm con un kit de síntesis de ARN tapado de acuerdo con las instrucciones del fabricante. Diluir el ARNm en 20 l de agua libre de RNasa y determinar la concentración. Alícuota de la ARN y almacenar a -80 ° C. </li><li> En el día de la inyección, preparar una solución de trabajo del ARNm con RNasa libre de agua estéril. Tenga en cuenta que 40 pg es generalmente la dosis ARN alta a la que no se producen efectos no específicos o tóxicos de la ARN. Mantenga la solución de trabajo en el hielo. Inyectar el embrión con MO y luego el ARNm de rescate, o co-inyectar los dos juntos. Por ejemplo, si la concentración de la prepar mRNAed en el paso 4,5 es de 0,6 mg / l, añadir 0,67 l de mRNA (0,4 g) a 4,33 l de RNasa libre de agua para hacer una concentración final de 0,08 mg / l, y luego una gota (500 pl) de cada inyección contiene 40 pg de mRNA. Preparar MO como se describe en el paso 3. </li></ol> 5. Preparación de embriones inyectados de Imagen Fluorescente <ol><li> Después de incubación a 28,5 ° CO / N, añadir 0,003% N-feniltiourea (PTU) al agua para evitar melanization E3, que afecta a la observación de la estructura pronefros mediante microscopía de fluorescencia. Sin embargo, no añada PTU antes de la gastrulación, ya que afecta el desarrollo embrionario temprano. </li><li> A las 48 HPF, dechorionate manualmente los embriones con pinzas finas. Tomar imágenes de 48 y 72 embriones HPF bajo un microscopio de disección luz. NOTA: Estas imágenes pueden ser tomadas sin anestesia. </li><li> Aproximadamente 10 min antes de la imagen bajo el microscopio de fluorescencia, anestesiar los embriones colocándolos enuna placa de Petri de 10 cm que contiene 160 mg / ml tamponada tricaína. Espere 5-10 minutos, y luego toque ligeramente el pez cebra para confirmar que no se mueven y por lo tanto la anestesia está trabajando. </li><li> Después de que los embriones son anestesiados, montarlos en el 3% de metilcelulosa y les orientan en posición prona con un microscopio de disección (por favor consulte la Tabla 1). </li><li> Observe la estructura pronefros bajo un microscopio de fluorescencia (por favor consulte la Tabla 1) y tomar fotografías a diferentes aumentos (10X y 20X). </li></ol>

<ol>
	<li>Choi, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdc42+deficiency+causes+ciliary+abnormalities+and+cystic+kidneys.">Cdc42 deficiency causes ciliary abnormalities and cystic kidneys.</a> J Am Soc Nephrol. 24 (9), 1435-1450 (2013).</li><li>Hostetter, C. L., Sullivan-Brown, J. L., Burdine, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+pronephros:+a+model+for+understanding+cystic+kidney+disease.">Zebrafish pronephros: a model for understanding cystic kidney disease.</a> Dev Dyn. 228 (3), 514-522 (2003).</li><li>Nyengaard, J. R., Bendtsen, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glomerular+number+and+size+in+relation+to+age,+kidney+weight,+and+body+surface+in+normal+man.">Glomerular number and size in relation to age, kidney weight, and body surface in normal man.</a> Anat Rec. 232 (2), 514-201 (1992).</li><li>Keller, G., Zimmer, G., Mall, G., Ritz, E., Amann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nephron+number+in+patients+with+primary+hypertension.">Nephron number in patients with primary hypertension.</a> N Engl J Med. 348 (2), 101-108 (2003).</li><li>Cullen-McEwen, L. A., Kett, M. M., Dowling, J., Anderson, W. P., Bertram, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nephron+number,+renal+function,+and+arterial+pressure+in+aged+GDNF+heterozygous+mice.">Nephron number, renal function, and arterial pressure in aged GDNF heterozygous mice.</a> Hypertension. 41 (2), 335-340 (2003).</li><li>Gerlach, G. F., Wingert, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney+organogenesis+in+the+zebrafish:+insights+into+vertebrate+nephrogenesis+and+regeneration.">Kidney organogenesis in the zebrafish: insights into vertebrate nephrogenesis and regeneration.</a> Wiley Interdiscip Rev Dev Biol. 2 (5), 559-585 (2013).</li><li>Igarashi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview:+nonmammalian+organisms+for+studies+of+kidney+development+and+disease.">Overview: nonmammalian organisms for studies of kidney development and disease.</a> J Am Soc Nephrol. 16 (2), 296-298 (2005).</li><li>Zhou, W., Boucher, R. C., Bollig, F., Englert, C., Hildebrandt, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+mesonephric+development+and+regeneration+using+transgenic+zebrafish.">Characterization of mesonephric development and regeneration using transgenic zebrafish.</a> Am J Physiol Renal Physiol. 299 (5), 1040-1047 (2010).</li><li>Liu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+defect+in+a+novel+Nek-family+kinase+causes+cystic+kidney+disease+in+the+mouse+and+in+zebrafish.">A defect in a novel Nek-family kinase causes cystic kidney disease in the mouse and in zebrafish.</a> Development. 129 (24), 5839-5846 (2002).</li><li>Slanchev, K., Putz, M., Schmitt, A., Kramer-Zucker, A., Walz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nephrocystin-4+is+required+for+pronephric+duct-dependent+cloaca+formation+in+zebrafish.">Nephrocystin-4 is required for pronephric duct-dependent cloaca formation in zebrafish.</a> Hum Mol Genet. 20 (16), 3119-3128 (2011).</li><li>Huang, L., et al. <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+Wnt5a+in+prostate+gland+development.">The role of Wnt5a in prostate gland development.</a> Dev Biol. 328 (2), 188-199 (2009).</li><li>Andre, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Wnt+coreceptor+Ryk+regulates+Wnt/planar+cell+polarity+by+modulating+the+degradation+of+the+core+planar+cell+polarity+component+Vangl2.">The Wnt coreceptor Ryk regulates Wnt/planar cell polarity by modulating the degradation of the core planar cell polarity component Vangl2.</a> J Biol Chem. 287 (53), 44518-44525 (2012).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203 (3), 253-310 (1995).</li><li>Torres, V. E., Harris, P. C., Pirson, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autosomal+dominant+polycystic+kidney+disease.">Autosomal dominant polycystic kidney disease.</a> Lancet. 369 (9569), 1287-1301 (2007).</li></ol>

The authors have nothing to disclose.

La enfermedad renal poliquística (PKD) es una de las principales causas de enfermedad renal terminal en los seres humanos y se caracteriza por la progresiva formación de quistes, agrandamiento renal y desarrollo de túbulos anormal 14. PKD autosómica dominante (PQRAD) es una enfermedad genética en la que la mutación de cualquiera de PKD1, que codifica policistina-1 (PC1), o PKD2, que codifica policistina-2 (PC2), los resultados en los riñones poliquísticos. Muchos otros genes, especialmente los que cod...

El pez cebra (Danio rerio), los embriones se han usado ampliamente como un modelo para estudiar el desarrollo del riñón y enfermedad renal poliquística. Hay muchas ventajas en el uso del pez cebra como modelo animal: la viabilidad de estudiar las interacciones genéticas, la capacidad de utilizar morfolinos antisentido (MO) durante caída de proteínas, la oportunidad para ensayar rápidamente un gran número de embriones, y la facilidad de ver fenotipos de órganos en larvas 1 viviente. Los pronefros es el primer riñón para desarrollar en los vertebrados y es funcional en larvas de pez cebra 2. La estructura de los pronefros pez cebra es relativamente simple en comparación con los metanefros de mamíferos, la tercera y última riñón a desarrollar en los mamíferos. La nefrona es la unidad de trabajo del riñón, con cada riñón humano contiene entre 500,000-1,000,000 nefronas 3,4 y cada riñón de ratón que tiene aproximadamente 13.000 nefronas 5, por lo que es difícil de observar la estructura nefrona única in riñones humanos o de ratón. El pez cebra tiene sólo dos nefronas y cada nefrona pez cebra contiene todos los componentes principales que se encuentran en el glomérulo y los túbulos de ratones y seres humanos 6 y tipos de células renales especializados similares. En comparación con otros modelos de vertebrados tales como Xenopus, la nefrona pez cebra se asemeja más a la nefrona de mamíferos, ya que tiene un sistema cerrado 7. En los últimos años, el genoma pez cebra ha sido secuenciado, lo que permite la amplia introducción de herramientas genéticas, amplios recursos de mutantes, y colecciones de líneas transgénicas reportero en modelos de pez cebra. Las formas pronefros pez cebra entre 12 a 72 h después de la fecundación (HPF) y pueden ser visualizados fácilmente en los embriones transparentes. La proteína del tumor de Wilms WT1 es un factor esencial para el desarrollo del riñón. Líneas de pez cebra transgénicos que expresan la proteína verde fluorescente (GFP) bajo el control del promotor wt1b Tg (wt1b: GFP) mostrar expre GFPsión encuentra específicamente en las regiones pronéfricos en embriones de pez cebra, a partir de 17 HPF 8. Nefronoptisis (NPHP), una enfermedad renal quística autosómica recesiva, es causada por mutaciones de genes NPHP 9. NPHP4 desmontables por morfolino causó la formación de quistes en la Tg (wt1b: GFP). Peces 10 Por lo tanto, este pez transgénico es un modelo adecuado para la observación de las estructuras del riñón y formación de quistes durante el desarrollo del riñón. Es importante destacar que la influencia de moduladores de desarrollo del riñón puede ser estudiada usando esta cepa en un tiempo y mano de obra de manera eficiente. Nuestro trabajo se describe el uso de Tg (wt1b: GFP) de pescado como un modelo para visualizar la formación de quistes renales después de la modulación de genes. Utilizamos inicio y empalme de sitio antisentido OM para derribar el gen Wnt5a en el pez cebra. Wnt5a es una glicoproteína secretada no canónica de la familia Wnt que juega un papel importante en el desarrollo de diversos órganos y postnatal celularfunción 11. Wnt5a trabaja a través de vías de Wnt no canónica, incluyendo la vía de la polaridad celular planar (PCP), que se ha encontrado a desempeñar un papel en la división celular orientado durante la elongación tubular renal. Wnt5a regula la vía / PCP Wnt mediante la formación de un complejo con el receptor tirosina quinasa (Ryk), que transduce más Wnt5a de señalización mediante la formación de un complejo con la proteína de la polaridad celular planar VANGL 2 (Vangl2), promoviendo así la estabilidad Vangl2 12. Los defectos en la vía de PCP pueden resultar en la división celular aleatoria y causar la formación de quiste renal. Utilizamos la Tg (wt1b: GFP) de la línea de pez cebra para observar la formación de quistes renales tras caída Wnt5a. La Tg (wt1b: GFP) modelo de pez cebra permite obtener imágenes en vivo y la observación puntual de la estructura del riñón. Después caída Wnt5a, formación de quistes renales se encontró a partir de las 24 HPF; en 72 hpf, los quistes se pueden encontrar en los glomérulos y los túbulos proximales. Este método también podría ser usado para sotros Creen genes que podrían causar la formación de quistes renales.

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			<td height="21" style="height:21px;width:221px;">0.5% Phenol-Red</td>
			<td style="width:229px;">Sigma</td>
			<td style="width:195px;">P0290</td>
			<td style="width:491px;">Color indicator for injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Morpholino</td>
			<td style="width:229px;">Genn Tools, LLC</td>
			<td style="width:195px;">(customized)</td>
			<td>Customized designed to the gene of interest</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">Pre-pulled needle</td>
			<td style="width:229px;">Tritech Research</td>
			<td style="width:195px;">MINJ-PP</td>
			<td style="width:491px;">If large amount of needle is required, you can also purchase a needle puller and prepare the needle in the lab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">T7 mMessage Kit</td>
			<td style="width:229px;">Ambion</td>
			<td style="width:195px;">1344</td>
			<td>For in vitro transcription to make capped mRNA for rescue experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">N-Phenylthiourea</td>
			<td style="width:229px;">Sigma</td>
			<td style="width:195px;">P7629</td>
			<td>For prevention of melanization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Tricaine</td>
			<td style="width:229px;">Sigma</td>
			<td style="width:195px;">A5040</td>
			<td>Also called ethyl 3-aminobenzoate, for zebrafish anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Methyl Cellulose</td>
			<td style="width:229px;">Sigma</td>
			<td style="width:195px;">M-0387</td>
			<td>For position of zebrafish&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">QIAquick PCR Purification Kit&#160;</td>
			<td style="width:229px;">Qiagen</td>
			<td style="width:195px;">28104</td>
			<td>For purification of PCR product for cap RNA synthesis.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">dNTP mix</td>
			<td style="width:229px;">Promega</td>
			<td style="width:195px;">U1511</td>
			<td>For PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Tag DNA Polymerase</td>
			<td style="width:229px;">Invitrogen</td>
			<td style="width:195px;">10342-053</td>
			<td>For PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;"></td>
			<td style="width:229px;"></td>
			<td style="width:195px;"></td>
			<td></td>
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			<td style="width:229px;"></td>
			<td style="width:195px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Equipment</td>
			<td style="width:229px;"></td>
			<td style="width:195px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">NanoDrop sepctrophotometer</td>
			<td style="width:229px;">Thermo Scientific</td>
			<td style="width:195px;">ND-1000</td>
			<td>For measure morpholino and RNA concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Air Compressor</td>
			<td style="width:229px;">Werther International, Inc.</td>
			<td style="width:195px;">Panther Compact 106</td>
			<td>Air source for injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Pico micro-injection pump</td>
			<td style="width:229px;">World Precision Instruments Inc</td>
			<td style="width:195px;">PV830 Pnematic PicoPump</td>
			<td>Other types of microinjection system can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Micro-manuplator</td>
			<td style="width:229px;">World Precision Instruments Inc</td>
			<td style="width:195px;">MMJR</td>
			<td>Right-handed (MMJL for left handed)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Needle holder</td>
			<td style="width:229px;">World Precision Instruments Inc</td>
			<td style="width:195px;">5430-ALL</td>
			<td>To hold needle for micromanipulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Dumont Tweezers</td>
			<td style="width:229px;">Fine Surgical Tools</td>
			<td style="width:195px;">11253-20</td>
			<td>For breaking off the needle tip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Dissecting microscope</td>
			<td>Leica M205C&#160;</td>
			<td style="width:195px;"></td>
			<td>For observing and imaging zebrafish embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Fluorscence microscope</td>
			<td>Zeiss Axio Obserer D1m &#160;</td>
			<td style="width:195px;"></td>
			<td>For imaging zebrafish pronephros</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Capillary tube (I.D 0.15 mm)</td>
			<td>VitroCom</td>
			<td style="width:195px;">CV1525Q-100</td>
			<td>For measure the volume of each injected drop</td>
		</tr>
	</tbody>
</table>

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.

Wnt5a derribar se logró mediante la introducción de traducción bloqueando MO (agosto-MO) o el exón / intrón empalme frontera MO (empalme-MO) a los embriones de pez cebra en la etapa de una célula. El AUG-MO se dirige al codón de inicio y, por lo tanto, inhibe tanto mensaje Wnt5a materna y cigóticos. El empalme de MO se dirige a la tercera sitio donador de empalme y sólo inhibe la transcripción cigóticos de Wnt5a (Figura 1A). La aumentación y morphants splice- phenocopied un...

Watch this Scientific Journal Video about A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys at JoVE.com

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 ...

a-possible-zebrafish-model-polycystic-kidney-disease-knockdown-wnt5a

Research

JoVE Journal

Medicine

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

11.0K vistas.  Eastern Virginia Medical School. 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. 

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

Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and teleost fish retain a remarkable capacity for cardiac regeneration with little or no scarring throughout life2,3. It is not known why only some non-mammalian vertebrates can recreate a complete organ from remnant tissues4,5. To understand the molecular and cellular differences between regenerative responses in different species, we need to use similar approaches for inducing acute injuries.
In mammals, the most frequently used model to study cardiac repair has been acute ischemia after a ligation of the coronary artery or tissue destruction after cryoinjury6,7. The cardiac regeneration in newts and zebrafish has been predominantly studied after a partial resection of the ventricular apex2,3. Recently, several groups have established the cryoinjury technique in adult zebrafish8-10. This method has a great potential because it allows a comparative discussion of the results obtained from the mammalian and non-mammalian species.
Here, we present a method to induce a reproducible disc-shaped infarct of the zebrafish ventricle by cryoinjury. This injury model is based on rapid freezing-thawing tissue, which results in massive cell death of about 20% of cardiomyocytes of the ventricular wall. First, a small incision was made through the chest with iridectomy scissors to access the heart. The ventricular wall was directly frozen by applying for 23-25 seconds a stainless steel cryoprobe precooled in liquid nitrogen. To stop the freezing of the heart, fish water at room temperature was dropped on the tip of the cryoprobe. The procedure is well tolerated by animals, with a survival rate of 95%.
To characterize the regenerative process, the hearts were collected and fixed at different days after cryoinjury. Subsequently, the specimen were embedded for cryosectioning. The slides with sections were processed for histological analysis, in situ hybridization and immunofluorescence. This undertaking enhances our understanding of the factors that are required for the regenerative plasticity in the zebrafish, and provide new insights into the machinery of cardiac regeneration. A conceptual and molecular understanding of heart regeneration in zebrafish will impact both developmental biology and regenerative medicine.

Zebrafish represents a valuable model to study the mechanisms of heart regeneration in vertebrates. Here, we present a protocol for induction of a heart infarct in adult zebrafish using cryoinjury. This method results in massive cell death within 20% of the ventricular wall, similar to that observed in mammalian infarcts.

La inducción de infarto de miocardio en adultos de pez cebra Uso de Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and ...

Investigación

Medicina

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Evaluación de los cambios teratogénicos en un modelo de pez cebra de la exposición fetal al alcohol

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

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

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

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 ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

La detección de Modificadores melanoma utilizando un modelo de pez cebra Tumor autóctona

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined.
We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.

Metabolic memory is the phenomenon by which diabetic complications persist and progress unimpeded even after euglycemia is achieved pharmaceutically. Here we describe a diabetes mellitus zebrafish model which is unique in that it allows for the examination of the mitotically transmissible epigenetic components of metabolic memory in vivo.

Un modelo de pez cebra de la diabetes mellitus y la memoria metabólica

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory ...

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 ...

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 ...

A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as cardiomyopathy. Because of its convenience and amenability to high throughput genetic manipulations, the generation of acquired cardiomyopathy models, such as the doxorubicin-induced cardiomyopathy (DIC) model in adult zebrafish, is opening the doors to new research avenues, including discovering cardiomyopathy modifiers via forward genetic screening. Different from the embryonic zebrafish DIC model, both initial acute and later chronic phases of cardiomyopathy can be determined in the adult zebrafish DIC model, enabling the study of stage-dependent signaling mechanisms and therapeutic strategies. However, variable results can be obtained with the current model, even in the hands of experienced investigators. To facilitate future implementation of the DIC model, we present a detailed protocol on how to generate this DIC model in adult zebrafish and describe two alternative ways of intraperitoneal (IP) injection. We further discuss options on how to reduce variations to obtain reliable results and provide suggestions on how to appropriately interpret the results.

A method to generate a doxorubicin-induced cardiomyopathy model in adult zebrafish (Danio rerio) is described here. Two alternative ways of intraperitoneal injection are presented and conditions to reduce variations among different experimental groups are discussed.

Un modelo de cardiomiopatía inducida por la doxorrubicina en pez cebra adulto

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as ...

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 ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Medición de la contractilidad isométrica de la arteria mesentérica de ratón con cable miografía

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...