Los autores agradecen a Adeline Dolly por las fotos de explante de fibronectina, Eric Theveneau útil para los debates y el Fondo para los Animales del Instituto Curie. CM es un becario postdoctoral de la Región Ile de France (Domaine d&#39;Interet Majeur vástago Pole), Université Paris Sud (Attache Temporaire d&#39;Enseignement et de Recherche) y la Agence Nationale de la Recherche. Este trabajo fue financiado por la Universite Paris Sud (Attractivite 2011), el Centre National de la Recherche Scientifique (Programa de Acción Thematique et incitativo sur), la Asociación pour la Recherche contre le cáncer de Grant SFI20101201882, Ligue contre le Cáncer, y la Agence Nationale de la Recherche (Agence Nationale de la Recherche Programa Blanc).

Los experimentos cumplen con la reglamentación nacional y europea relativa a la protección de los animales utilizados para fines científicos y con los principios establecidos a nivel internacional de sustitución, reducción y refinamiento. 1. Preparación de platos recubiertos con fibronectina 14 <ol><li> Pipetear 500 ml de 10 mg / ml de fibronectina de grado cultivo diluido en 1x tampón fosfato salino (PBS) en una placa de Petri estándar de plástico (Ø 40 x 11 mm). Incubar a 37 ° C durante 1 hora. Nota: si usa platos de cristal o cubreobjetos de vidrio para la imagen posterior, utilice una solución de fibronectina 100 mg / ml. </li><li> Enjuague tres veces con PBS 1x. </li><li> Reemplazar PBS con 500 ml de 1x PBS/0.1% albúmina de suero bovino (BSA). Incubar a 37 ° C durante 30 min. </li><li> Enjuague tres veces con PBS 1x. </li><li> Llenar con 1x PBS y se mantenga a 4 ° C hasta su uso (por lo general de 24 a 48 h). </li></ol> 2. La disección de los explantes NC &lt;ol&gt; <li> Preparar 3/4 normal Anfibio Medio (NAM) (NaCl 110 mM, 2 mM de KCl, 1 mM de Ca (NO3) 2, 1 mM de MgSO4, 0,1 mM EDTA, 1 mM de NaHCO3, 0,2 x PBS, 50 mg / ml gentamicina 10,15). No guardar más de 1 semana para la disección, se almacenan a 4 ° C. NAM Mayor se puede utilizar para la preparación de platos de disección de agarosa recubiertas con (abajo). </li><li> Preparar 60 mm placas de Petri para la disección, recubiertas con 10 ml de agarosa al 2% en 3/4 de NAM. </li><li> Recoger herramientas de disección: pipetas Pasteur de plástico, una mejor pasador de insecto montado sobre soporte de pasador (ver tabla materiales para la referencia), un &quot;cuchillo de la ceja&quot; (hecho con pelo de las cejas incrustado en parafina en la punta de una pipeta Pasteur de vidrio 16), dos fina pinza de disección, pipeta P1000 y consejos, un estereoscopio binocular con aumento de 8-40X y 50, una fuente de luz brillante con fibras ópticas guías. Todas las herramientas de disección deben mantenerse limpios, después de cada experimento, enjuagarlos dos veces con agua destilada, una vez with etanol al 100% y dejar secar. Almacene lejos del polvo. </li><li> Llene el plato de disección con NAM fresca casi hasta la cima. </li><li> Obtener X. laevis embriones de acuerdo con los procedimientos estándar 10 y ellos envejecer hasta que alcanzan Néurula etapa 16 (de acuerdo con Nieuwkoop y Faber mesa de desarrollo 17). </li><li> Coloque etapa 16 embriones en el plato de disección utilizando la pipeta Pasteur de plástico. </li><li> Llevar a cabo todos los pasos subsiguientes en el marco del alcance binocular. Retire la membrana vitelina con dos pares de pinzas. Tenga cuidado de no dañar la parte dorsal de los embriones. </li><li> Espere hasta que los embriones han llegado a la etapa 17 para iniciar la disección. Etapa 17 es apropiado, ya que la cresta neural está claramente separado del tubo neural, en contraste con las etapas anteriores. Después de la etapa 17, se inicia la migración de Carolina del Norte y mezclándose con el mesénquima circundante. </li><li> Cavar un agujero pequeño en la agarosa con pinzas para hacer un hueco para los embriones con el fin de mantenerdurante la disección. Coloque un embrión dorsal hacia arriba. Nota: el modelado de la arcilla puede ser usado para mantener los embriones en lugar de excavación de un agujero en la agarosa. </li><li> Hacer una incisión en la parte lateral del pliegue neural anterior con el cuchillo de la ceja y el pasador de insectos. Tenga cuidado de no ir demasiado profundo en el embrión: tratar de cortar a través de 2-3 capas de células (nunca llegar tan profundo como el archenteron!). Durante todo el proceso de disección, nunca permitir que los tejidos disecados para entrar en contacto con la interfaz aire-líquido desde X. tejidos embrionarios laevis se lisan por la tensión superficial. </li><li> Haga una segunda incisión paralela en la parte más dorsal del pliegue neural utilizando los mismos instrumentos. </li><li> Si es necesario, gire el plato alrededor de 90 °. Hacer tercera incisión perpendicular con el cuchillo de la ceja y la clavija de insecto, en el lado posterior de las dos primera incisiones. La NC es una gruesa masa de células por debajo de la capa externa pigmentada del ectodermo, lateralpara el tubo neural. </li><li> Utilice la punta de la cuchilla de cejas para separar la capa de ectodermo pigmentada de la NC subyacente, a partir de la tercera borde de la incisión. </li><li> Utilice el lado de la cuchilla de cejas para separar el tejido NC a partir de la tercera incisión. El tejido NC es más bien gris, mientras que el mesodermo cefálico subyacente es blanco. Separe con cuidado la Carolina del Norte a partir del mesodermo. </li><li> Separe el tejido NC anterior de la vesícula óptica, y cortar en este nivel para liberar el explante. Como control para la primera experimentos, comprobando la expresión Snail2 (un marcador canónica Carolina del Norte) en algunos explantes por hibridación in situ se recomienda. </li></ol> 3. Plating explantes de fibronectina <ol><li> Rellena el plato fibronectina recubierto con Modificada Danilchick Medio (MDM, NaCl 53 mM, Na2CO 3 5 mM, K Gluconato 4,5 mM, Na Gluconato 32 mM, MgSO4 1 mM, CaCl2 1 mM, BSA al 0,1%, se ajusta el pH a 8.3 con 1 M Bicina <sup&gt; 18). </li><li> La transferencia de los explantes en el plato fibronectina recubierto con una pipeta P1000. Nunca permita que los explantes para tocar la interfase aire-líquido. En primer lugar, la pipeta un poco de líquido en la punta, a continuación, los explantes, a continuación, algunos de nuevo líquido. No permita que el aire en la punta durante la transferencia de los explantes en el plato recubiertos de fibronectina. Una vez que la punta está dentro del líquido del plato fibronectina recubiertos, dejar que los explantes lentamente bajan por gravedad, o empujar con mucha suavidad. Evitar la expulsión de los explantes demasiado rápido. </li><li> Coloque 10-30 explantes en el plato con el cuchillo de la ceja. Evitar los lados del plato, que no son adecuados para la formación de imágenes posterior. </li><li> Trate de no mover el plato hasta que los explantes se han unido a la fibronectina (se tarda unos 15-30 minutos). Si es necesario, mueva el plato con mucho cuidado. </li><li> Coloque el plato en un incubador refrigerado (entre 15-20 º C, para la tabla de temperaturas ver 17). Cuando se coloca a 15 ° C, las células are suele migrar de manera eficiente y dispersando después de 6 horas de incubación (el tiempo puede variar entre los experimentos). </li></ol> 4. Injerto explantes en embriones de acogida <ol><li> Cortar un cubreobjetos de vidrio fina en piezas muy pequeñas utilizando fórceps gruesas. El tamaño de las piezas debe ser de aproximadamente 1,5 mm 2. Sumergir los trozos en una placa de Petri que contiene de 3/4 NAM o PBS usando fórceps. </li><li> Preparación del embrión de acogida: diseccionar la NC anfitrión en 3/4 NAM como se describe en 3) con especial cuidado de no dañar el embrión de acogida mientras se quita la membrana vitelina y la disección de la NC. La tensión del tejido tiende a aumentar el tamaño de la ablación: dejar que el embrión de acogida curar parcialmente, mientras que la disección de la embrión donante. </li><li> En otro plato de agarosa recubiertas con o en otra parte del mismo plato, diseccionar el explante NC del embrión donante como se describe en el Protocolo 3. Transferir el injerto de Carolina del Norte en el plato que contiene el host con cuidado, como se describe enProtocolo 3. </li><li> Colocar inmediatamente el explante NC a la zona de ablación host utilizando fórceps. El explante debe adjuntar parcialmente. Ponga un pedazo de cubreobjetos de vidrio sobre el embrión injertado para mantener el injerto en su lugar. Elige un pedazo de vidrio más grande que el embrión para evitar dañarla. El embrión será un poco aplanada. </li><li> Espere por lo menos 15 a 20 minutos, luego retire suavemente el cubreobjetos. Deje que el embrión se recuperan durante otros 10 minutos y la pipeta con cuidado en un plato limpio lleno de 3/4 NAM, utilizando la pipeta Pasteur de plástico. </li></ol>

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B., Hanken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Runx2+is+essential+for+larval+hyobranchial+cartilage+formation+in+Xenopus+laevis.">Runx2 is essential for larval hyobranchial cartilage formation in Xenopus laevis.</a> Dev. Dyn. 236 (6), 1650-1662 (2007).</li><li>Yan, C. Y. I., Skourides, P., Chang, C., Samba Brivanlou, 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+Xenopus+hnRNP+expressed+in+neural+and+neural+crest+tissues.">a Xenopus hnRNP expressed in neural and neural crest tissues.</a> Dev. Dyn. 238 (1), 204-209 (2009).</li><li>Acloque, H., Adams, M. S., Fishwick, K., Bronner-Fraser, M., Nieto, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+transitions:+the+importance+of+changing+cell+state+in+development+and+disease.">Epithelial-mesenchymal transitions: the importance of changing cell state in development and disease.</a> J. Clin. Invest. 119 (6), 1438-1449 (2009).</li></ol>

Los autores no tienen nada que revelar.

Este protocolo describe una técnica fácil de explante premigratorio craneal NC en embriones de X. laevis. Los embriones utilizados para dichos experimentos deben ser robustos y sanar bien. Deseche cualquier lote de embriones saludables. Además, crecer los embriones a diferentes temperaturas (de 12 a 18-20 ° C), con el fin de escindir la cresta neural en la etapa 17 y no más tarde. Después de la etapa 17, la cresta neural se puede mezclar con mesodermo cefálico y no se puede eliminar completamente. Tejido...

La cresta neural (NC) es una población de células embrionarias transitoria que emerge del tubo neural en el extremo del tubo neural en embriones de vertebrados. Los eventos de señalización y genéticos que controlan la especificación NC comenzar tan pronto como la gastrulación. La NC se especifica en la frontera entre el ectodermo neural y no neural por las señales de los tejidos que rodean dorsales. Al final de la neurulación, NC células se someten a una transición epitelio-mesénquima (EMT) y migran extensamente en el embrión siguientes rutas estereotipadas de responder a las señales de guía circundante. Una vez que han llegado a su destino final, se diferencian en una amplia gama de derivados, por ejemplo, neuronas, células gliales, huesos, cartílagos y células pigmentadas 1-5. Debido a su contribución a muchos tipos de células y tejidos embrionarios, defectos en cualquier trámite del desarrollo de células NC, desde la inducción de la diferenciación final, puede causar síndromes congénitos con nombre neurocristopathies 6. Emanipulación Xperimental del NC en desarrollo en diferentes etapas - de especificación, EMT, migración y diferenciación - mejorará nuestra comprensión de neurocristopathies y permitir el diseño de estrategias terapéuticas potenciales. El embrión de Xenopus laevis es un modelo de elección para estudiar el desarrollo de NC. Un gran número de embriones son fáciles de obtener, y la fertilización externa da acceso a las primeras etapas de desarrollo. Muchas herramientas están disponibles para manipular experimentalmente X. laevis desarrollo embrionario. Ganancia de función y derribo Gene son fáciles de realizar mediante la microinyección de células individuales de primeros blástulas. Tejidos embrionarios pueden ser cortados para la reasociación in vitro o ensayos de 7-11 back-injerto 12,13. En este protocolo, se describe cómo diseccionar premigratorio NC craneal en X. laevis neurulas tarde, antes de la migración. Estos explantes se pueden cultivar en fibronectina-Coated placas para estudiar la migración y la diferenciación en controladas en condiciones experimentales in vitro. Explantes NC también se pueden injertar en embriones normales o manipuladas de acogida para estudiar su migración y diferenciación in vivo.

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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Fibronectin from bovine plasma</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">F4759-1MG</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Bovine Serum Albumine. Fraction V</td>
			<td style="width:71px;">Euromedex</td>
			<td style="width:119px;">04-100-811-C</td>
			<td>Lower quality grade may&#160; be suitable for this application</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Stainless Steel Insect Pins. Size 000</td>
			<td style="width:71px;">FST</td>
			<td align="right" style="width:119px;">26001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Dumont #5 forceps 0.05 mm x 0.02 mm</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">11252-20</td>
			<td></td>
		</tr>
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dissection of xenopus laevis neural crest for in vitro explant culture or in vivo transplantation

La cresta neural (NC) es una población de células dorsal del tubo neural transitoria que sufre una transición epitelio-mesénquima (EMT) al final de la neurulación, migra ampliamente hacia diversos órganos, y se diferencia en muchos tipos de derivados (neuronas, neuroglia, cartílago y hueso, células pigmentadas y endocrinos). En este protocolo, se describe la forma de diseccionar la premigratorio craneal NC a partir de embriones de Xenopus laevis, con el fin de estudiar el desarrollo de NC in vivo e in vitro. El modelo de la rana ofrece muchas ventajas para estudiar el desarrollo temprano; abundantes lotes están disponibles, los embriones se desarrollan rápidamente, ganancia in vivo y la pérdida de las estrategias de función permiten la manipulación de la expresión génica antes de la disección NC en donante y / o embriones de acogida. Los explantes de NC se pueden sembrar en fibronectina y utilizados para estudios in vitro. Ellos pueden cultivarse durante varios días en un medio definido libre de suero. También describe cómo injertar explantes NC espaldaen embriones de acogida para el estudio de NC migración y diferenciación in vivo.

Cuando chapada en fibronectina, los explantes de la cresta neural se adhieren rápidamente (15-30 min) y el explante se extiende plana dentro de 2 horas (Figura 1A). Después de 3-6 células hr comienzan a dispersarse. En 24 horas a 15 ° C, muchas células han comenzado a migrar fuera del explante (figuras 1B y 1C). Yema hace que las células muy brillante bajo contraste de fase (Figura 1B). Protuberancias celulares (filopodes y lamellipodes) son clara...

Watch this Scientific Journal Video about Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation at JoVE.com

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

Este protocolo describe cómo diseccionar la cresta neural craneal premigratorio (NC) de neurulas Xenopus laevis. Estos explantes se pueden sembrar en fibronectina y cultivadas in vitro, o injertados en embriones de acogida. Esta técnica permite el estudio de los mecanismos de la NC transición epitelio-mesénquima, la migración y la diferenciación.

La disección de<em&gt; Xenopus laevis</em&gt; Neural Crest para<em&gt; In vitro</em&gt; Explante cultura o<em&gt; In vivo</em&gt; Trasplante

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of ...

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Research

JoVE Journal

Biology

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

13.1K vistas.  Centre Universitaire.  Este protocolo describe cómo diseccionar la cresta neural craneal premigratorio (NC) de neurulas Xenopus laevis. Estos explantes se pueden sembrar en fibronectina y cultivadas in vitro, o injertados en embriones de acogida. Esta técnica permite el estudio de los mecanismos de la NC transición epitelio-mesénquima, la migración y la diferenciación. 

Video: Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.

La disección del Organizador y explantes Animal Polo de Xenopus laevis embriones y de la Asamblea de un ensayo de adhesión celular

Investigación

Biología

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

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

La obtención de huevos de Xenopus laevis mujeres

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Preparación y fraccionamiento de los extractos de huevo Xenopus laevis

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Microinyección de oocitos de Xenopus laevis

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

Determinación del tejido Con el trasplante de Cap Animal (ACT) en el ensayo Xenopus laevis</em

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

La producción de transgénicos Xenopus laevis Integración mediada por la enzima de restricción y transferencia nuclear

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

Patch clamp y técnicas de perfusión para el estudio de los canales iónicos expresados ​​en Xenopus Ovocitos

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

Los trasplantes faciales en<em&gt; Xenopus laevis</em&gt; Los embriones

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.