Los autores agradecen a Leif Oxburgh y Derek Adams por compartir generosamente sus conocimientos, Leif Oxburgh por los útiles comentarios sobre el manuscrito, y Stefan Wölfl y Ulrike Müller por su apoyo técnico y Saskia Schmitteckert, Julia Gobbert, Sascha Weyer y Viola Mayer por su ayuda en el laboratorio. Este trabajo fue apoyado por Desarrollo, La Empresa de Biólogos (a CP).

Los ratones se mantuvieron de acuerdo con la normativa sueca y la legislación de la Unión Europea (2010/63 / UE). Todos los procedimientos se realizaron siguiendo las directrices del Comité Sueco de Ética (permisos C79 / 9, C248 / 11, y C135 / 14). El Regierungspräsidium Karlsruhe y los Oficiales de Bienestar Animal de la Universidad de Heidelberg han aprobado procedimientos en la Universidad de Heidelberg sobre animales. 1. Preparación de reactivos y materiales para la cultura NOTA: Use una campana de flujo laminar para minimizar la contaminación. <ol><li> En el día de la disección, la Fc humana previa a la agrupación o la proteína de efrina quimérica recombinante se fusionaron a Fc humano mezclándola con anticuerpos de cabra Fc antihumanos en una proporción molar de 1: 5 en solución salina tamponada con fosfato estéril (PBS). Incubar durante 1 h a 37 ° C 5 . </li><li> Preparar el medio de cultivo suplementando Dulbecco&#39;s Modified Eagle Medium: NutrientMezcla F-12 (DMEM / F12) con 1% de penicilina / estreptomicina (v / v), 1% de glutamina (v / v), 1% de insulina-transferrina-selenio (v / v) Transferrina y 1,5 μg / ml de anfotericina. NOTA: 10 ml de medio son suficientes para 16 embriones. </li><li> Añada una solución concentrada de efrina recombinante a una concentración final de 20 μg / ml al medio de cultivo. Utilice Fc humano agrupado como control. </li><li> Preparar las placas de cultivo añadiendo 500 μl de medio de cultivo a cada pocillo de una placa estéril de 4 pocillos de poliestireno de fondo plano sin recubrimiento. </li><li> Usando fórceps estériles, coloque cuidadosamente un filtro de membrana de policarbonato con un tamaño de poro de 5 μm por pocillo en la parte superior del medio. Transferir la placa preparada a la incubadora (37 ° C / 5% CO 2 ). Asegúrese de que el filtro permanezca seco en la superficie superior y flote. NOTA: Un filtro es suficiente para dos anlagen de riñón. </li><li> Utilizando una cuchilla de afeitar, corte aproximadamente 30 puntas de pipeta (volumen: 100 μl) aprox.Y 1 mm de la punta para dar lugar a una abertura más amplia. Coloque las puntas de nuevo en un rack pipette-tip. </li></ol> 2. Disección de Rudimentos Metanefricos en E11.5 <ol><li> Sacrifique a un ratón embarazado cronometrado en E11.5 por dislocación cervical (o usando un método aprobado por el comité de ética local). </li><li> Retirar el útero y los embriones, según lo descrito por Brown et al 6 . A partir de este punto, trabajar bajo un microscopio de disección. </li><li> Usando la pinza de relojería No. 5, corte el embrión directamente debajo de las patas delanteras. Utilice un plato de petri fresco para cada embrión. Quitar las piernas y la cola del embrión. Para ello, agarra el tejido que se va a quitar con un par de fórceps relojero nº 5 y usa las puntas de un segundo par para cortar. </li><li> Para retirar la masa del órgano ventral, use las puntas de un par de pinzas para abrir lateralmente la pared del cuerpo del embrión en una dirección rostral a caudal. <ol><li> Corte superficialmente, paralelo a tLa aorta dorsal. Utilice la aorta dorsal llena de sangre y por lo tanto visible para la orientación. Separar cuidadosamente la parte ventral de la pared del cuerpo de la parte dorsal usando las puntas de la pinza cerrada y voltear el embrión. Corte la pared del cuerpo lateralmente en una dirección rostral a caudal y usando la aorta dorsal para la orientación, como para el lado anterior. </li></ol></li><li> Una vez que la pared del cuerpo ventral se ha separado del cuerpo dorsal, agárralo con un par de fórceps y tire con cuidado para quitarlo, junto con la masa del órgano. NOTA: El metanéfrico anlagen suele permanecer unido a la pared del cuerpo dorsal. Son estructuras ovales de ~ 200 μm de largo situadas al nivel de las extremidades posteriores. </li><li> Sujete la pared del cuerpo dorsal con un par de fórceps, con el lado ventral hacia arriba. Utilizando el otro par de pinzas, agarrar la aorta dorsal en su extremo rostral y cuidadosamente pelar la aorta dorsal de la pared del cuerpo dorsal. NOTA: Ambos riñones metanefricos generalmente se mantienen unidosA la aorta dorsal. </li><li> Usando un par de fórceps, corte rostralmente al riñón anlagen para eliminar la aorta. Luego, cuidadosamente separar los dos anlagen renal utilizando las puntas de la pinza. </li><li> Cuidadosamente quitar el tejido no deseado del riñón anlagen con las puntas de la pinza. Tenga cuidado de no dañar el mesénquima, ya que el daño puede resultar en un pobre crecimiento y la exclusión del explante de un análisis posterior. </li><li> Transferir el anlagen renal por separado con una pipeta de microlitros y una punta de pipeta abierta en 10 μL de PBS al filtro de membrana de policarbonato en una placa de 4 pocillos. NOTA: Debido a la tensión superficial, el explante se cubrirá con una fina capa de medio. Un filtro es suficiente para dos anlagen renal. <ol><li> Coloque cada uno de los dos anlagen renal en el centro de cada mitad respectiva del filtro. Transferir la placa de nuevo a la incubadora a 37 ° C / 5% de CO 2 . Dejar la placa en la incubadora hasta que el ron metanefricImentes del siguiente embrión están listos para ser colocados en el filtro. </li></ol></li><li> Repita los pasos 2.3-2.9.1 para cada embrión. NOTA: Con cierta práctica, el procedimiento de disección tomará aproximadamente 5 min por embrión. </li><li> Incubar el riñón anlagen durante 3 días a 37 ° C / 5% de CO 2 . </li></ol> 3. Preparación de reactivos para fijación y tinción <ol><li> Para preparar paraformaldehído (PFA) al 4% en PBS sin calcio y magnesio (p / v), disolver el PFA a 60 ° C en PBS, agitando continuamente. Enfriar a temperatura ambiente y utilizar un filtro de papel para eliminar las partículas restantes. Alícuota y mantener a 4 ° C para su uso inmediato o congelar para el almacenamiento a largo plazo. PRECAUCIÓN: La PFA es tóxica. Use el equipo de protección personal especificado en las normas de seguridad locales y trabaje en una campana extractora de humos. Deseche los desechos siguiendo las pautas institucionales y usando contenedores de desecho designados. </li><li> Para preparar la solución de permeabilización, diluir Triton X-100En PBS sin calcio y magnesio hasta 0,3% (v / v). </li><li> Para preparar la solución de bloqueo, añadir 5% (v / v) de suero de cabra y 0,1% Triton X-100 a PBS sin calcio y magnesio. Añadir azida sódica hasta una concentración final de 0,02% (p / v). Almacenar a 4 ° C. PRECAUCIÓN: La azida sódica es tóxica. Manipular la azida sódica de acuerdo con las normas locales de seguridad y protección del medio ambiente. NOTA: Cuando se usan anticuerpos para la tinción, use suero al 5% de la especie de la que se derivó el anticuerpo secundario para hacer la solución de bloqueo. </li><li> Diluir la aglutinina de Dolichorus biflorus biotinilada 1: 200 en solución de bloqueo. Diluir Alexa488-streptavidin conjugado 1: 200 en solución de bloqueo. </li><li> Para preparar un medio de incrustación listo para usar (véase la tabla de materiales), añadir 0,1 g de mucoadhesivo / ml a base de alcohol polivinílico soluble en agua a glicerol al 25% (p / v) en Tris-HCl 0,1 M (pH 8,5) . Calentar a 50 ° C bajo agitación continua durante 1 h, enfriar y ajustar el pH a 8,0-8,5 usando 1M de NaOH. Evitar mayores concentraciones de NaOH. Añadir 100 μg / ml de N-propilgalato y timerosal hasta una concentración final de 0,02% (p / v). Se agita durante 30 minutos a temperatura ambiente. PRECAUCIÓN: El timerosal es tóxico. Manipularlo de acuerdo con las normas locales de seguridad y protección del medio ambiente. <ol><li> Llenar el medio de inclusión en tubos de 50 ml, equilibrar el rotor y centrifugar a 3.200 xg ya temperatura ambiente durante 10 min. Decantar la solución transparente en tubos de 15 mL y desechar el gránulo. Alícuota y almacenar a -20 ° C durante varias semanas. Una vez descongelado, mantener la solución a 4 ° C. Caliente a ~ 30 ° C inmediatamente antes de usar. </li></ol></li><li> Prepare tantos portaobjetos de vidrio como los filtros con explantes deben montarse. Para ello, pegue dos pequeños cubreobjetos, 18 x 18 mm, a cada lado de la diapositiva de vidrio con adhesivo instantáneo, Deje un espacio de ~ 15 mm para el filtro entre. </li></ol> 4. Fijación y tinción <ol><li> Después de un período de cultivo de 3Días in vitro (div), retirar las placas de la incubadora y aspirar cuidadosamente el medio de cultivo de cada pocillo usando una micropipeta. Asegúrese de no tocar el filtro y mantenga la punta abierta hacia la pared del pozo. </li><li> Añadir 500 μl de PFA al 4% / PBS a cada pocillo. Los filtros flotarán sobre el fijador. Usando una micropipeta, agregue cuidadosamente el fijador PFA gota a gota para sumergir los filtros. Incubar a temperatura ambiente durante 30 min. NOTA: Para todos los pasos siguientes, se debe tener cuidado de no lavar los explantes del filtro. Mantenga la abertura de la punta de la pipeta hacia la pared del pozo. </li><li> Retire el PFA y enjuague dos veces con 500 μl de PBS, sumergiendo el filtro. Añadir 500 μl de solución de permeabilización a cada pocillo e incubar a temperatura ambiente durante 1 h. </li><li> Enjuagar dos veces con 500 μL de PBS y añadir 500 μL de solución de bloqueo a cada pocillo. Incubar a temperatura ambiente durante 1 h. </li><li> Reemplace la solución de bloqueo por250 μl de aglutinina biotinilada Dolichorus biflorus diluida 1: 200 en solución de bloqueo e incubar a 4 ° C durante 24 h. </li><li> Retire la solución de aglutinina de Dolichorus biflorus y enjuague dos veces con 500 μl de PBS por pocillo. </li><li> Añadir 250 μL de estreptavidina conjugada con Alexa488 diluida 1: 200 en solución de bloqueo e incubar a 4 ° C durante 24 h. Alternativamente, incubar a temperatura ambiente durante 2 h. NOTA: Se consigue una mejor relación señal / ruido cuando se incuba a 4 ° C. </li><li> Enjuague dos veces con 500 μl de PBS por pocillo. Usando fórceps, transfiera los filtros a las diapositivas de vidrio preparadas, con los explantes hacia arriba. Montar con ~ 50-100 μl de medio de incrustación. Cubrir con cubreobjetos No. 1.5 de 60 mm de largo. Dejar que la solución del medio de incrustación se solidifique durante 2 h a temperatura ambiente en la oscuridad. Proceda a la formación de imágenes o almacene las diapositivas, envueltas en papel de aluminio, a 4 ° C hasta que esté listo para la imagen. </li><li> Imagen con un microscopio de epifluorescencia de campo ancho 7 &lt;/ Sup&gt; acoplado a una lámpara de Hg y utilizar una unidad de espejo para la excitación azul (paso de banda de excitación, 460 - 495 nm, espejo dicromático, 505 nm y paso de banda de emisión, 510 - 550 nm) y 20X Plan - Apochromat, 0,75 numérico abertura. </li></ol>

<ol>
	<li>Sax&#233;n, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organogenesis of the kidney. Developmental and Cell Biology Series. 19, (1987).</li><li>Lindstr&#246;m, N. O., 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=Integrated+&#946;-catenin,+BMP,+PTEN,+and+Notch+signalling+patterns+the+nephron.">Integrated &#946;-catenin, BMP, PTEN, and Notch signalling patterns the nephron.</a> eLife. 4, e04000 (2015).</li><li>Peuckert, C., 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=Multimodal+Eph/Ephrin+signaling+controls+several+phases+of+urogenital+development.">Multimodal Eph/Ephrin signaling controls several phases of urogenital development.</a> Kidney Int. 90 (2), 373-388 (2016).</li><li>Pasquale, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eph+receptor+signalling+casts+a+wide+net+on+cell+behaviour.">Eph receptor signalling casts a wide net on cell behaviour.</a> Nat Rev Mol Cell Biol. 6 (6), 462-475 (2005).</li><li>Bonanomi, D., 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=Ret+Is+a+Multifunctional+Coreceptor+that+Integrates+Diffusible-+and+Contact-Axon+Guidance+Signals.">Ret Is a Multifunctional Coreceptor that Integrates Diffusible- and Contact-Axon Guidance Signals.</a> Cell. 148 (2), 568-582 (2012).</li><li>Brown, A. C., 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=Isolation+and+Culture+of+Cells+from+the+Nephrogenic+Zone+of+the+Embryonic+Mouse+Kidney.">Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney.</a> J Vis Exp. (50), e2555 (2011).</li><li>Grobstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inductive+interaction+in+the+development+of+the+mouse+metanephros.">Inductive interaction in the development of the mouse metanephros.</a> J Exp Zool. 130, 319-340 (1955).</li><li>Sax&#233;n, L., Toivonen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Primary Embryonic Induction. , (1962).</li><li>Sax&#233;n, L., Koskimies, O., Lahti, A., Miettinen, H., Rapola, J., Wartiovaara, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+kidney+mesenchyme+in+an+experimental+model+system.">Differentiation of kidney mesenchyme in an experimental model system.</a> Adv Morphog. 7, 251-293 (1968).</li><li>Dudley, A. T., Godin, R. E., Robertson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+FGF+and+BMP+signaling+pathways+regulates+development+of+metanephric+mesenchyme.">Interaction between FGF and BMP signaling pathways regulates development of metanephric mesenchyme.</a> Genes Dev. 13, 1601-1613 (1999).</li><li>Per&#228;l&#228;, N., 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=Sema4C-Plexin+B2+signalling+modulates+ureteric+branching+in+developing+kidney.">Sema4C-Plexin B2 signalling modulates ureteric branching in developing kidney.</a> Differentiation. 81 (2), 81-91 (2011).</li><li>Thesleff, I., Ekblom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+transferrin+in+branching+morphogenesis,+growth+and+differentiation+of+the+embryonic+kidney.">Role of transferrin in branching morphogenesis, growth and differentiation of the embryonic kidney.</a> J Embryol exp Morph. 82, 147-161 (1984).</li><li>Watanabe, T., Costantini, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+analysis+of+ureteric+bud+branching+morphogenesis.">Real-time analysis of ureteric bud branching morphogenesis.</a> Dev Biol. 271, 98-108 (2004).</li><li>Sebinger, D. D. R., Unbekandt, M., Ganeva, V. V., Ofenbauer, A., Werner, C., Davies, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel,+Low-Volume+Method+for+Organ+Culture+of+Embryonic+Kidneys+That+Allows+Development+of+Cortico-Medullary+Anatomical+Organization.">A Novel, Low-Volume Method for Organ Culture of Embryonic Kidneys That Allows Development of Cortico-Medullary Anatomical Organization.</a> PLoS One. 5 (5), e10550 (2010).</li><li>Ekblom, P., Miettinen, A., Virtanen, I., Wahlstr&#246;m, T., Dawnay, A., Sax&#233;n, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+segregation+of+the+metanephric+nephron.">In vitro segregation of the metanephric nephron.</a> Dev Biol. 84 (1), 88-95 (1981).</li><li>Davies, J. A., Unbekandt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=siRNA-mediated+RNA+interference+in+embryonic+kidney+organ+culture.">siRNA-mediated RNA interference in embryonic kidney organ culture.</a> Methods Mol Biol. 886, 295-303 (2012).</li><li>Sax&#233;n, L., Lehtonen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+kidney+in+organ+culture.">Embryonic kidney in organ culture.</a> Differentiation. 36 (1), 2-11 (1987).</li><li>Bard, J. B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+the+mouse+kidney+embryogenesis+writ+small.">The development of the mouse kidney embryogenesis writ small.</a> Curr Opin Genet Dev. 2, 589-595 (1992).</li><li>Davies, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+cold+storage+and+transport+of+viable+embryonic+kidney+rudiments.">A method for cold storage and transport of viable embryonic kidney rudiments.</a> Kidney Int. 70 (11), 2031-2034 (2006).</li><li>Batourina, E., 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=Distal+ureter+morphogenesis+depends+on+epithelial+cell+remodeling+mediated+by+vitamin+A+and+Ret.">Distal ureter morphogenesis depends on epithelial cell remodeling mediated by vitamin A and Ret.</a> Nat Genet. 32 (1), 109-115 (2002).</li></ol>

Los autores no tienen nada que revelar.

Este manuscrito describe un método para aislar el desarrollo de metanfric anlagen del embrión de ratón y para cultivar los órganos rudiments. Este método es una técnica estándar, desarrollada por Grobstein 8 y Saxén 9 , 10 , y fue adaptada y modificada por muchos otros 11 , 12 . El éxito del método depende principalmente de la duración de la disección, ya que la superv...

The mammalian kidney is derived from two primordial structures with mesodermal origin: the tubular epithelial ureteric bud and the metanephric mesenchyme. During nephrogenesis, the ureteric bud invades the metanephric mesenchyme and branches to form the collecting system. The metanephric mesenchyme gives rise to the epithelial elements of the nephrons. These processes occur in a precisely timed and spatially coordinated manner and are initiated by reciprocal inductive mechanisms. Both tissue components communicate and affect the other's cell morphogenesis. 
In the 1920s, it was Boyden who performed the in vivo obstruction of the mesonephric duct in chicken, providing the first indication of inductive interactions as separated nephric blastema fail to differentiate1. At about the same time, the first successful attempts to culture chicken nephric rudiments in a hanging drop were published. Subsequently, the organ culture was developed to study tissue interactions in mammalian organogenesis. In the 1950s, Grobstein developed a technique in which metanephric rudiments could be cultured on a filter. This technique was modified by Sax&#233;n, who placed the filter on a Trowell-type screen in a culture dish1. Over the years, many modifications and applications for organ culture have emerged. The method described here is based on Sax&#233;n's technique but is simplified, as the filters float free on the medium and the diameter of the culture well only slightly exceeds the diameter of the filter, limiting unwanted movement of the filter.
Whole-organ culture is a classical, cheap, and simple but powerful tool to investigate cellular processes and intercellular communication during organogenesis. Organ culture allows for treatment with biological agents, such as growth factors, antibodies, antisense oligonucleotides, viruses, and peptides, as well as with pharmaceutical compounds and other chemicals. Also, gene function may be studied using explants derived from genetically modified mice or using inducible gene inactivation technology, such as the Cre-loxP system. This allows for the study of genetic mutations that cause embryonic lethality prior to the development of the kidney. Organ culture can also be combined with fluorescent tagging for gene function or lineage tracing and modern imaging techniques, which enable real-time monitoring of cell behavior2.
In the specific example provided here, the effect of EphrinB2-activated Eph-receptor signaling on the branching morphology of the ureteric bud was investigated. The morphology of the EphA4/EphB2 double-knockout mice suggested several severe defects in kidney development, which were detectable as early as embryonic day 11 (E11) and involved the ureteric bud, the ureter, and the common nephric duct3. Signaling via Eph receptors requires the clustering of the ligand-receptor dimer4. To over-activate Eph signaling, the kidney rudiments from E11.5 mouse embryos were cultured in the presence of clustered recombinant EphrinB2-Fc. EphrinB2 is a known ligand for the EphA4 receptor, which is expressed in the ureteric bud tips3.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM/F-12</td>
			<td>Thermo Fisher Scientific</td>
			<td>21331020</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140148</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, calcium, magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>14040117</td>
			<td>use for dissection</td>
		</tr>
		<tr>
			<td>holo-Transferrin human</td>
			<td>Sigma-Aldrich</td>
			<td>T0665</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium (ITS -G) (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>41400045</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B solution</td>
			<td>Sigma-Aldrich</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S8032</td>
			<td></td>
		</tr>
		<tr>
			<td>Thimerosal</td>
			<td>Sigma-Aldrich</td>
			<td>T5125</td>
			<td></td>
		</tr>
		<tr>
			<td>Propyl gallate</td>
			<td>Sigma-Aldrich</td>
			<td>2370</td>
			<td></td>
		</tr>
		<tr>
			<td>Mowiol 4-88</td>
			<td>Sigma-Aldrich</td>
			<td>81381</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated Dolichorus Biflorus Agglutinin</td>
			<td>Vector Laboratories</td>
			<td>B-1035</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa488 conjugated Streptavidin</td>
			<td>Jackson Immuno Research</td>
			<td>016-540-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse Ephrin-B2 Fc Chimera Protein, CF</td>
			<td>R&#38;D Systems</td>
			<td>496-EB</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IgG1 Fc, CF</td>
			<td>R&#38;D Systems</td>
			<td>110-HG-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Human IgG Fc Antibody</td>
			<td>R&#38;D Systems</td>
			<td>G-102-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline tablets</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td>use for fixation and immunostaining</td>
		</tr>
		<tr>
			<td>Dumont #5, biologie 
			tips, INOX, 11cm</td>
			<td>agnthos.se</td>
			<td>0208-5-PS</td>
			<td>2 pairs of forceps are needed</td>
		</tr>
		<tr>
			<td>Iris scissors, straight, 12cm</td>
			<td>agnthos.se</td>
			<td>03-320-120</td>
			<td></td>
		</tr>
		<tr>
			<td>Dressing Forceps, 
			straight, delicate, 13cm</td>
			<td>agnthos.se</td>
			<td>08-032-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes Nunclo Delta treated</td>
			<td>Thermo Fisher Scientific</td>
			<td>150679</td>
			<td></td>
		</tr>
		<tr>
			<td>TMTP01300 Isopore Membrane Filter, polycarbonate, Hydrophilic, 5.0 &#181;m, 13 mm, white, plain</td>
			<td>MerckMillipore</td>
			<td>TMTP01300</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunclon Multidishes 
			4 wells, flat bottom</td>
			<td>Sigma-Aldrich</td>
			<td>D6789-1CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glass24x50mm thickn. No.1.5H 0.17+/-0.005mm</td>
			<td>nordicbiolabs</td>
			<td>107222</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glasses No.1.5, 18x18mm</td>
			<td>nordicbiolabs</td>
			<td>102032</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides ~76x26x1, 1/2-w. ground plain</td>
			<td>nordicbiolabs</td>
			<td>1030418</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Razor Blades</td>
			<td>VWR</td>
			<td>55411-055</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tubes</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430828</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430055</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman prepleated qualitative filter paper, Grade 113V, creped</td>
			<td>Sigma-Aldrich</td>
			<td>WHA1213125</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixed stage research mircoscope</td>
			<td>Olympus</td>
			<td>BX61WI</td>
			<td></td>
		</tr>
		<tr>
			<td>Black 6 inbred mice, male, C57BL/6NTac</td>
			<td>Taconic</td>
			<td>B6-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Black 6 inbred mice,female, C57BL/6NTac</td>
			<td>Taconic</td>
			<td>B6-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Greenough Stereo Microscope</td>
			<td>Leica</td>
			<td>Leica S6 E</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection and culture of mouse embryonic kidney

El objetivo de este protocolo es describir un método para la disección, aislamiento y cultivo de rutinas metanefricas de ratón. Durante el desarrollo del riñón de los mamíferos, los dos tejidos progenitores, el brote ureteral y el mesénquima metanéfrico, se comunican y inducen recíprocamente mecanismos celulares para formar eventualmente el sistema colector y los nefrones del riñón. A medida que los embriones de mamíferos crecen intrauterinos y por lo tanto son inaccesibles para el observador, se ha desarrollado un cultivo de órganos. Con este método, es posible estudiar las interacciones epiteliales-mesenquimales y el comportamiento celular durante la organogénesis renal. Además, se puede investigar el origen de las malformaciones congénitas del riñón y del tracto urogenital. Después de una disección cuidadosa, los rudimentos metanefricos se transfieren a un filtro que flota sobre el medio de cultivo y puede mantenerse en una incubadora de cultivo celular durante varios días. Sin embargo, hay que tener en cuenta que las condiciones sonArtificial y podría influir en el metabolismo en el tejido. Además, la penetración de sustancias de ensayo podría estar limitada debido a la matriz extracelular y la membrana basal presentes en el explante. Una ventaja principal de la cultura de órganos es que el experimentador puede obtener acceso directo al órgano. Esta tecnología es barata, sencilla y permite un gran número de modificaciones, como la adición de sustancias biológicamente activas, el estudio de variantes genéticas y la aplicación de técnicas de imagen avanzada.

El anlagen renal metanéfrico se obtuvo a partir de ratones endocrinos Black-6 preñados a E11.5 y se cultivaron. Después de 3 días, la yema ureterica se había ramificado hasta 5 veces, dando como resultado una ramificación de la yema ureterica inicialmente en forma de T. Se fotografió cada explante y se cuantificaron los números de segmentos y puntos finales para determinar las generaciones de ramificación y para calcular el número de puntos finales por rama ( Figura 1</...

Watch this Scientific Journal Video about Dissection and Culture of Mouse Embryonic Kidney at JoVE.com

Dissection and Culture of Mouse Embryonic Kidney

Este protocolo describe un método para aislar y cultivar rudimentos metanefricos de embriones de ratón.

Disección y cultivo de riñón embrionario de ratón

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

dissection-and-culture-of-mouse-embryonic-kidney

Research

JoVE Journal

Developmental Biology

11.8K vistas.  Uppsala University.  Este protocolo describe un método para aislar y cultivar rudimentos metanefricos de embriones de ratón. 

Video: Dissection and Culture of Mouse Embryonic Kidney

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

A gran escala de pez cebra embrionario del corazón Disección de Análisis transcripcional

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

Investigación

Biología del Desarrollo

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Cultura de embriones de ratón explantes cocleares y Gene Transfer por electroporación

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

Cultura y Co-cultura de ratón ovarios y los folículos ováricos

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

Diferenciación neural de células madre embrionarias de ratón en el suero libre de monocapa Cultura

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Transcriptoma de perfiles de<em&gt; In-Vivo</em&gt; Productos fabricados preimplantación de embriones bovinos mediante plataforma de microarrays de dos colores

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

Cultura de murinos embrionarias Metatarsianos: Un modelo fisiológico de osificación endocondral

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate embryonic origins and functions &#8212; the neurohypophysis and the adenohypophysis. The developing mouse pituitary gland is tiny and delicate with an elongated oval shape. A coronal section is preferred to display both the adenohypophysis and neurohypophysis in a single slice of the mouse pituitary.
The goal of this protocol is to achieve proper pituitary coronal sections with well-preserved tissue architectures from developing mice. In this protocol, we describe in detail how to dissect and process pituitary glands properly from developing mice. First, mice are fixed by transcardial perfusion of formaldehyde prior to dissection. Then three different dissecting techniques are applied to obtain intact pituitary glands depending on the age of mice. For fetal mice aged embryonic days (E) 17.5 - 18.5 and neonates up to 4 days, the entire sella regions including the sphenoid bone, gland, and trigeminal nerves are dissected. For pups aged postnatal days (P) 5 - 14, the pituitary glands connected with trigeminal nerves are dissected as a whole. For mice over 3 weeks old, the pituitary glands are carefully dissected free from the surrounding tissues. We also display how to embed the pituitary glands in a proper orientation by using the surrounding tissues as landmarks to obtain satisfying coronal sections. These methods are useful in analyzing histological and developmental features of pituitary glands in developing mice.

We present a protocol to dissect pituitary glands and prepare pituitary coronal sections from developing mice.

Disección y preparación del segmento Coronal de desarrollo de hipófisis de ratón

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

Disección y la coloración de los ovarios Pupal de Drosophila

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

Cultura Organotypic método para estudiar el desarrollo de tejidos de embrión de pollo

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

This protocol describes the dissection and culture of cranial neural crest cells from mouse models, primarily for the study of cell migration. We describe the live imaging techniques used and the analysis of speed and cell shape changes.

Disección, cultivo y análisis de células de cresta neural craneal primarias de ratón para el estudio de la delaminación y migración de células de cresta neuronal

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ...