Este trabajo es apoyado por una beca de la MRC al señor FS.

1. Desarrollo de los embriones <ol><li> Cuando se establecen, los huevos están agrupados por filamentos de fijación en el corion. Para permitir que los embriones se desarrollan normalmente, es necesario separar los huevos. Maraña y cortar los filamentos apego mediante la celebración de los filamentos de unión con dos pinzas. </li><li> Vez deshecho el clúster, los huevos se separan de las heces y las algas y la transfieren a un medio de embriones frescos, con una densidad máxima de 40 huevos por placa de Petri de 6 cm. </li><li> Embriones medaka desarrollo algo más lento que el pez cebra a 27 ° C. El tiempo de incubación es diferente entre las dos especies; embriones medaka escotilla del corion en 7 días y de inmediato empezar a nadar y comer, mientras que embriones de pez cebra se incuban en 2 días, pero empezar a nadar y comer en 5-6 días. Desarrollo de medaka se clasifican de acuerdo a cuatro puesta en escena de Iwamatsu. </li><li> El desarrollo de los embriones medaka puede ser convenientemente ajustada a los planes experimentales mediante la selección de la temperatura adecuada. Desarrollo de los embriones medaka se puede detener a 4 ° C en el desarrollo temprano. Después de la etapa 24, cuando empieza a corazón latiendo, el desarrollo puede ser frenado con una temperatura mínima de 18 ° C. </li><li> El momento de aparición de órganos / tejidos es ligeramente diferente en medaka comparación con el pez cebra, es decir, en somitogenesis medaka se produce después del inicio del desarrollo del cerebro, mientras que en el pez cebra somitegenesis precede el desarrollo del cerebro. </li></ol> 2. Extracción del corion El corion de medaka se compone de dos capas de protección con una capa dura interna y una superficie exterior suave. Por lo tanto, un tratamiento de la proteasa en dos pasos empleando la enzima pronasa y la eclosión es necesario eliminar esta corion. Una vez dechorionated, los embriones deben mantenerse en 1X BSS. Semi-estériles condiciones mejorarán el éxito del cultivo de embriones dechorionated, especialmente a partir de períodos de observación son necesarios. Estos incluyen el uso de soluciones estériles (por ejemplo, esterilizados 1X BSS con antibióticos) y herramientas esterilizadas con etanol al 70% seguido de un enjuague con 1X BSS. Como dechorionated embriones medaka son más suaves y más frágiles que los embriones de pez cebra dechorionated, cuidado se debe tomar para asegurarse de que no entren en contacto con el aire o burbujas en la pipeta, ya que esto provocaría un colapso inmediato. Para asegurar un mínimo daño a los embriones, una pipeta de calor de boca ancha de vidrio pulido con una bomba de pipeta se debe utilizar para la transferencia de embriones y un lazo del pelo deben ser utilizados para orientar los embriones para la observación. No adherente placas de Petri se deben utilizar para evitar que los embriones se adhieran a las superficies. <ol><li> Antes de dechorionation se debe comprobar que los huevos han sido lo suficientemente separados y limpiados. </li><li> Dado que tanto pronasa y enzimas proteasas para incubar, deben mantenerse en hielo y minimizar la exposición de los embriones de estas enzimas. </li><li> La transferencia de los huevos de papel de lija (P2000 tamaño de grano, resistente al agua) colocado en la tapa de una caja de Petri de 9 cm. Retire el exceso de medio, sino garantizar un volumen suficiente como para evitar que restos de secado de los embriones. </li><li> Suavemente gire en torno a los embriones de 45 a 60 segundos para eliminar algunos de los pelos y la superficie externa ligeramente marcas en la superficie del corion (Figura 1). La transferencia de embriones back to original placa de Petri y examinar. Cuando los embriones que rueda sobre el papel de lija usar el dedo índice, aplicando una presión mínima y manteniendo los dedos en paralelo a la superficie de papel de lija. No ruede más de 5.7 embriones a la vez. Esta precaución reducirá al mínimo el riesgo de aplastamiento de los embriones por debajo de la otra. </li><li> Vuelva a colocar medio huevo en un plato con pronasa 20mg/ml y los embriones se incuban durante 40-60 minutos a 27 ° C. Asegúrese de que los embriones son suficientemente cubierto por una tapa pronasa y está presente en el plato. Pronasa no utilizada debe ser mantenido en hielo durante este paso para evitar la auto-digestión y se puede reutilizar hasta que la actividad se pierde (aproximadamente 1-2 semanas). </li><li> Recuperar para su reutilización y pronasa embriones lavar 5 veces en medio de embriones para eliminar los restos de pronasa, ya que esto inactivar la enzima para incubar añadió. </li><li> Quitar medio de embriones y embriones de la cubierta con la enzima de la eclosión, garantizando los embriones se siente como una monocapa en el plato. Si los huevos se sientan encima de uno al otro en el plato, los de abajo serán aplastados como el corion se disuelve. Mantenga las enzimas utilizadas para incubar en hielo. </li><li> Incubar los embriones a los 27 ° C y después de 15 minutos compruebe periódicamente el avance de la eclosión usando un microscopio estereoscópico. Se verá que una serie de cráter lunar-como los agujeros comienzan a aparecer en la capa interna del corion, que pronto se disuelve después de este salir de la suave capa externa del corion quitar fácilmente manually. Todo el proceso de incubación puede tomar 15-60 minutos. </li><li> Tan pronto como los embriones salen del corion, la transferencia de embriones a una placa de Petri que contiene 1X BSS. Asegúrese de no transferir la enzima para incubar a la BSS 1X tocando la punta de la pipeta sobre la superficie de la BSS permite que los embriones a rodar con cuidado. </li><li> Una vez que todos los embriones son transferidos de la enzima de la eclosión, hacer una transferencia definitiva a otro plato fresco de 1X BSS. Esto asegura que los embriones no están expuestos a los restos de la enzima de la eclosión, ya que podría dañar los embriones expuestos. Una vez en el plato final, los embriones siguen poseyendo la capa externa del corion puede ser liberado manualmente utilizando unas pinzas esterilizadas micro-bajo el microscopio estereoscópico. Si los embriones deben ser desarrollados dechorionation siguiente, a la penicilina / estreptomicina, debe añadirse a la SEV para prevenir el crecimiento bacteriano. </li></ol><img src="/files/ftp_upload/2055/2055fig1.jpg" alt="Figura 1" /> Figura 1. Comparación de los embriones durante el enrollado y desenrollado dechorionation. Observe cómo los embriones rodó en el panel B carecen de los pelos se ve en los embriones desenrolla en el panel A. 3. Montaje de embriones dechorionated Incorporación de agarosa es útil para períodos más largos de la imagen (por ejemplo, time-lapse de imágenes) de embriones vivos, así como de las observaciones detalladas de embriones fijo. Durante la gastrulación y organogénesis temprana (etapa 14 a la etapa 28), los embriones medaka exhiben ondas rítmicas de los movimientos de contracción a través de la peridermis, una capa de tejido que cubre tanto el desarrollo del embrión y la yema de huevo 5. Los embriones son tratados con 3,5 mm 1-heptanol de detener los movimientos de contracción 6. Para detener el movimiento de embriones después de la etapa 28 (64hpf), los embriones son anestesiados mediante la adición de gotas de tricaína mesilato (TMS) antes de la incrustación. TMS también se agrega a la agarosa (añadir una cantidad mínima, esta dosis debe ser optimizado, de aproximadamente una dilución de 1:25). <ol><li> Descongelar una cantidad pequeña de baja temperatura de gelificación del 3% de agarosa (en 1X BSS) por calentamiento a 37 º C y mantener a esta temperatura. </li><li> Los siguientes pasos deben llevarse a cabo con rapidez para garantizar que la agarosa no se solidifique antes de orientar el embrión. Mediante una transferencia de vidrio de boca ancha pipeta de agarosa fundida suficiente para llenar la tapa de la depresión de un tubo de Eppendorf. </li><li> La transferencia de un embrión dechorionated a la depresión tapa minimizar llevar más de BSS con el embrión. Inmediatamente la absorción de la agarosa y el embrión de la depresión de la tapa y la transferencia a una cámara de cultivo placa de Petri. Una placa de Petri de 3,5 cm con una ventana de cristal en la parte inferior se utiliza. Para obtener imágenes mediante un microscopio invertido, los embriones son orientados hacia abajo, el cuerpo y se coloca cerca de la cubierta de vidrio. Para obtener imágenes con un microscopio vertical, el espesor de agarosa se reduce al mínimo. </li><li> La transferencia de la placa de Petri de 3,5 cm de diámetro en una placa de Petri que contiene 14 cm de hielo y agua (lleno de agua a la profundidad de aproximadamente 1 / 3 del total de plato grande). Mientras mantiene la cámara firmemente en la parte inferior de la placa de Petri 14 cm, utilice un bucle de cabello suavemente para orientar el embrión como se desee en la agarosa fundida y retener el embrión hasta que la agarosa solidifica suavemente subiendo y bajando el plato más pequeño a su posición original en el agua helada. </li></ol> 4. El trasplante de células de embriones medaka El objetivo de este procedimiento es determinar si el gen de interés actúa en células de manera autónoma (dentro de una célula) o no de células de forma autónoma (entre las células). Las células del donante puede ser etiquetada con un tinte trazador como rodamina dextrano antes del trasplante (como se describe en &#39;La microinyección de embriones de medaka &quot;el protocolo que acompaña Jove) o una cepa transgénica con la expresión de GFP se puede utilizar, permitiendo una evaluación del trasplante. Una combinación de ambas técnicas de etiquetado a menudo es útil para superar la auto-fluorescencia que se pueden encontrar. Para los estudios de lapso de tiempo después del trasplante, la expresión de GFP es particularmente útil. Use una pipeta de vidrio de boca ancha con una bomba de pipeta a través de este procedimiento y esterilizar todas las herramientas (incluyendo diapositivas) de antemano con EtOH al 70% seguido por un enjuague con agua estéril BSS 1X. Receptor embriones se desarrollan normalmente en torno a la 12 ª etapa, ya que permite la discriminación de los polos ventral y dorsal al llevar a cabo el trasplante. <ol><li> Comenzará dechorionating embriones 1,5 horas antes del trasplante y aparatos de inyección de configuración durante la incubación enzimática. </li><li> Colocar un portaobjetos de microscopio cavidad en un diámetro de 9 cm de placa de Petri. </li&gt; <li> Agregue una pequeña cantidad del 3% de metilcelulosa en el centro de la diapositiva cavidad con una punta de pipeta estériles y diluirse. </li><li> Metilcelulosa en seco durante unos 1-2 minutos. </li><li> Agregar 350μl de BSS estéril 1X para llenar la depresión de diapositivas. </li><li> La transferencia de un embrión donante y un máximo de cuatro embriones receptores a la diapositiva utilizando una pipeta de vidrio. </li><li> Embriones orientarse mediante un bucle de cabello por lo que el blastodermo del embrión es hacia arriba. Los embriones se pueden inclinó unos contra otros para aumentar la estabilidad adicional. </li><li> Inserte suavemente la micro-aguja en blastodermo donantes y poco a poco la absorción de 10-20 células. </li><li> Suavemente inserte la aguja en el área requerida de blastodermo receptores embrión y expulsar a las células lentamente. Gran cuidado se debe tomar cuando se inserta en las células del blastodermo beneficiarias a fin de no interrumpir las células de la yema de los límites del saco, ya que puede provocar la muerte del embrión. </li><li> Vierta esterilizados 1X BSS en la placa de Petri. Vierta cuidadosamente la BSS en el plato lo más cerca posible del lado de la antena como sea posible a fin de no interrumpir los embriones. Nunca vierta el BSS directamente sobre los embriones y añadir suficiente BSS de forma que la diapositiva y los embriones se encuentran inmersos. </li><li> Agregar 100μl de penicilina / estreptomicina al plato y la tapa. Con cuidado, llevar la cápsula a una incubadora de 27 ° C para permitir el desarrollo normal. </li><li> Los embriones pueden ser observadas periódicamente como se desee. Cuando se utiliza para llevar a cabo el trasplante de ganancia de función y / o experimentos fenotipo de rescate, algunos de los cambios morfológicos observados podrían deberse a los efectos del trasplante. Así, los trasplantes múltiples son necesarios. Después de 2-3 días, melanóforos debe estar presente en el saco vitelino, cabeza, ojos y tronco. Si está presente en los embriones trasplantados luego un éxito quimera ha sido probablemente producida (Figura 3). </li></ol> Si es necesario, el genotipo del embrión donante (s) por la transferencia de PCR de tubo (s) con 25μl de 20mg/ml proteinasa K. Se incuba a 55 ° C durante 4 horas seguido de 10 minutos a 94 ° C y llevar a cabo la PCR. 5. Los resultados representativos <img src="/files/ftp_upload/2055/2055fig2.jpg" alt="Figura 2" /> Figura 2. Posición Ejemplo de un embrión de agarosa embebidos. Anterior se muestra a la derecha y la vista es dorsal. Grupo B: las células endoteliales se puede ver a ambos lados del cuerpo del embrión y se etiquetan con las buenas prácticas agrarias impulsadas por el promotor fli. <img src="/files/ftp_upload/2055/2055fig3.jpg" alt="Figura 3" /> Figura 3. Imágenes de los embriones después del trasplante. La imagen A muestra un embrión de donante y el receptor inmediato post-trasplante. El embrión donante es de la derecha con un blastodermo completamente rojo (flecha). El embrión destinatario se muestra a la izquierda y se identifica fácilmente por la pequeña masa de rojo las células trasplantadas visible en el blastodermo (cabeza de flecha). La imagen B muestra dos embriones receptores de aproximadamente 7 horas post-trasplante (se incuba a 27 ° C). Observe que las células trasplantadas migraron desde el sitio del trasplante y ahora están dispersos por todo el blastodermo (flechas). La imagen C muestra un embrión receptor en st.29 (aproximadamente 74hpf). Tenga en cuenta la pigmentación en los ojos y la presencia de melanóforos en la región del tronco y el cerebro (flechas). Las células rodamina-etiquetados puede ser visto como la colonización muchas de las estructuras embrionarias que indica el éxito de la producción de una quimera.

<ol>
	<li>Furutani-Seiki, M., Sasado, T., Morinaga, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+genome-wide+screen+for+mutations+affecting+organogenesis+in+Medaka,+Oryzias+latipes.">A systematic genome-wide screen for mutations affecting organogenesis in Medaka, Oryzias latipes.</a> Mech Dev. 121, 647-658 (2004).</li><li>Furutani-Seiki, M., Wittbrodt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medaka+and+zebrafish,+an+evolutionary+twin+study.">Medaka and zebrafish, an evolutionary twin study.</a> Mech Dev. 121, 629-6237 (2004).</li><li>Hirose, Y., Varga, Z. M., Kondoh, H., Furutani-Seiki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+lineage+and+regionalization+of+cell+populations+during+Medaka+neurulation.">Single cell lineage and regionalization of cell populations during Medaka neurulation.</a> Development. 131, 2553-2563 (2004).</li><li>Iwamatsu, T. <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+normal+development+in+the+medaka+Oryzias+latipes.">Stages of normal development in the medaka Oryzias latipes.</a> Zool Sci. 11, 825-839 (1994).</li><li>Cope, J., Fluck, R., Nicklas, L., Plumhoff, L. A., Sincock, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stellate+layer+and+rhythmic+contractions+of+the+Oryzias+latipes+embryo.">The stellate layer and rhythmic contractions of the Oryzias latipes embryo.</a> J Exp Zool. 254, 270-275 (1990).</li><li>Brend, T., Holley, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+Whole+Mount+High-Resolution+Double+Fluorescent+In+Situ+Hybridization.">Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization.</a> J Vis Exp. , (2009).</li></ol>

En este video se muestra cómo dechorionate y llevar a cabo transplanataion celular en embriones medaka. Esta es una poderosa técnica para producir embriones quiméricos para el estudio de la función de genes / proteínas durante el desarrollo, así como para aclarar la autonomía de los genes / proteínas en cuestión. Medaka son un sistema de vertebrados modelo útil para esta técnica debido a los mapas de destino bien establecido y sus préstamos a la transparencia y en vivo imágenes en tiempo real. El trasplante puede ser combinado con microinyección (como se describe en &#39;La microinyección de embriones medaka &quot;el protocolo que acompaña Jove) para que el comportamiento de las células trasplantadas se pueden observar fácilmente.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Embryo medium</td>
<td>Reagent</td>
<td>Made in-house</td>
<td>N/A</td>
<td>200ml 50X stock solution, 1ml 1%methyleneblue in H2O/10 liter RO water (50X stock solution: NaCl 14.7g, KCl 0.6g, CaCl2 2H2O 2.4g, MgSO4 7H2O 4.0g/1 litre RO water.)</td>
</tr>
<tr>
<td>Micro-dissecting forceps</td>
<td>Tools</td>
<td>55 INOX A.DUMONT&amp;FILS</td>
<td>N/A</td>
<td>Very sharp fine tips for removal of chorion.</td>
</tr>
<tr>
<td>Fine waterproof sandpaper - p2000 grit size</td>
<td>Tools</td>
<td>Hermes Abrasives Ltd.</td>
<td>N/A</td>
<td>Very fine grit size for rolling/cleaning embryos with chorions.</td>
</tr>
<tr>
<td>3% methylcellulose</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M 0512</td>
<td>High viscosity for holding embryos in place during transplantation.</td>
</tr>
<tr>
<td>1X BSS</td>
<td>Reagent</td>
<td>Made in-house</td>
<td>N/A</td>
<td>20XBSS: 130g NaCl, 8g KCl, 4g MgSO4.7H2O, 4g CaCl2.2H2O, and 10mg Phenol Red in 1L MilliQ water and autoclave; 500mM Hepes in MilliQ water autoclaved. Add 25ml 20XBSS and 15ml 500ml Hepes pH7.0, fill up to 500ml and filter sterilize before use.</td>
</tr>
<tr>
<td>Penicillin-streptomycin</td>
<td>Reagent</td>
<td>Gibco</td>
<td>15140-122</td>
<td>Added to BSS when incubating dechorionated embryos.</td>
</tr>
<tr>
<td>Pronase</td>
<td>Reagent</td>
<td>Calbiochem</td>
<td>53702</td>
<td>For dechorionation.</td>
</tr>
<tr>
<td>Hatching enzyme</td>
<td>Reagent</td>
<td>Made in-house</td>
<td>N/A</td>
<td>For dechorionation.</td>
</tr>
<tr>
<td>Tricaine mesilate (TMS)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-5040</td>
<td>400mg tricaine in 97.9ml distilled water and 2.1ml 1M Tris, pH adjusted to 7; 4.2ml of this solution in 100ml clean tank water.</td>
</tr>
<tr>
<td>3% ultra-low gelling temp. agarose</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-2576</td>
<td>Type IX-A. Used during embedding of embryos for observation.</td>
</tr>
<tr>
<td>Petri dish culture chamber</td>
<td>Tool</td>
<td>Iwaki, Asahi Techno Glass Corp.</td>
<td>11-004-008</td>
<td>Iwaki glass base dish 35mm with 12mm window. Used during embedding of embryos for observation.</td>
</tr>
<tr>
<td>Micromanipulator</td>
<td>Equipment</td>
<td>Narishige</td>
<td>N/A</td>
<td>Model MN151. For removal and insertion of cells during transplantation.</td>
</tr>
<tr>
<td>Borosilicate glass capillaries</td>
<td>Tool</td>
<td>Harvard Apparatus</td>
<td>GC100-10T</td>
<td>For making transplantation needles.</td>
</tr>
<tr>
<td>Flaming/Brown micropipette puller</td>
<td>Equipment</td>
<td>Sutter Instrument Co.</td>
<td>Model P-97 </td>
<td>For making transplantation needles.</td>
</tr>		</tbody>
		</table>
		</div>

dechorionation of medaka embryos and cell transplantation for the generation of chimeras

Medaka es una pequeña puesta de huevos de peces de agua dulce que permite a los análisis genéticos y embriológicos y es uno de los tres organismos modelo vertebrado en el que todo el genoma impulsado fenotipo mutante pantallas se llevaron a cabo 1. Divergencia de superposición de funciones de los genes relacionados entre medaka y pez cebra permite la identificación de los fenotipos de la novela que no son identificables en una sola especie 2, por lo tanto medaka y pez cebra son complementarios para la disección genética de las funciones genoma de los vertebrados. Manipulación de embriones medaka, como dechorionation, los embriones de montaje de imagen y el trasplante de células, son procedimientos claves para trabajar en ambos medaka y pez cebra en un laboratorio. El trasplante de células examina la autonomía de las mutaciones de células medaka. Las quimeras son generados mediante el trasplante de células marcadas a partir de embriones de donantes en los embriones receptores sin etiquetar. Las células del donante se pueden transplantar a zonas específicas de los embriones receptores basados ​​en el destino mapas 3 de modo que los clones de las células trasplantadas se pueden integrar en el tejido de interés durante el desarrollo. Debido a que el corion duro y blando de embriones, la manipulación de embriones medaka es más complicado que en el pez cebra. En este vídeo, se muestran los procedimientos detallados para manipular embriones medaka.

Watch this Scientific Journal Video about Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras at JoVE.com

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Debido a que el corion duro y blando de embriones, la manipulación de embriones medaka es más complicado que en el pez cebra. Este video muestra paso a paso los procedimientos para la forma de manipular embriones medaka, incluyendo dechorionation, montaje de agarosa para la imagen y el trasplante de células para la producción de quimeras. Estos procedimientos son esenciales para el uso medaka y pez cebra en un laboratorio para sacar el máximo provecho de sus características complementarias para la disección genética de las funciones de genoma de los vertebrados.

Dechorionation de embriones de medaka y trasplante de células para la generación de quimeras

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in ...

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17.6K vistas.  University of Bath. Debido a que el corion duro y blando de embriones, la manipulación de embriones medaka es más complicado que en el pez cebra. Este video muestra paso a paso los procedimientos para la forma de manipular embriones medaka, incluyendo dechorionation, montaje de agarosa para la imagen y el trasplante de células para la producción de quimeras. Estos procedimientos son esenciales para el uso medaka y pez cebra en un laboratorio para sacar el máximo provecho de sus caracter...

Video: Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

Aislamiento y trasplante de células madre hematopoyéticas (HSC)

Investigación

Biología

Tracheotomy: A Method for Transplantation of Stem Cells to the Lung

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell therapy may alleviate many of the symptoms associated with lung disease and induce alveolar repair in adults. Concurrent with the ongoing search for stem cells applicable for human treatment, precise delivery and homing (to the site of disease) must be reassured for successful therapy. Here, I report that stem cells can safely be instilled via the trachea opening a non-stop route to the lung. This method involves a skin incision, caudal insertion of a cannula into and along the tracheal lumen, and injection of a stem cell vehicle mixture into airways of the lung. A broad range of media solutions and stabilizers can be instilled via tracheotomy, resulting in the ability to deliver a wider range of cell types. With alveolar epithelium confining these cells to the lumen, lung expansion and negative pressure during inhalation may also assist in stem cell integration. Tracheal delivery of stem cells, with a quick uptake and the ability to handle a large range of treatments, could accelerate the development of cell-based therapies, opening new avenues for treatment of lung disease.

Significant breakthroughs in stem cell identification are continuously being made. To translate these discoveries, however, novel methods for cellular delivery must be devised. Here I report that the airways provide a safe route for stem cell transplantation to the lungs.

Traqueotomía: Un método para el trasplante de células madre para el pulmón

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell ...

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

Plástico inclusión y corte de Xenopus laevis embriones

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

Generación de humanos CD40 activado por células B

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

La generación de embriones quiméricos de pez cebra mediante un trasplante

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

Tejido diana quimeras embrionarias: pez cebra Gástrula Trasplante de células

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Preparación de embriones para Microscopía Electrónica de la Drosophila Tubo cardíaco embrionario

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

El trasplante de las buenas prácticas agrarias-que expresa Blastómeros de imágenes en vivo del desarrollo de la retina y el cerebro en embriones de pez cebra quiméricos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

Microinyección de embriones de medaka para su uso como un organismo modelo genético

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generación de ratones derivados de células madre pluripotentes inducidas

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Methods Article

Dechorionation de embriones de medaka y trasplante de células para la generación de quimeras