Os autores agradecem Adeline Dolly para as fotos de explante sobre a fibronectina, Eric Théveneau para discussões úteis e Biotério do Institut Curie. CM é um pós-doutorado da Região Ile de France (Domaine d&#39;Interet Majeur Stem Pole), Universite Paris Sud (Adido Temporaire d&#39;Enseignement et de Recherche) e Agence Nationale de la Recherche. Este trabalho foi financiado pela Universite Paris Sud (Attractivite 2011), o Centre National de la Recherche Scientifique (Programa de Acção Thématique et incitativo sur), Associação pour la Recherche contre le Câncer Grant SFI20101201882, Ligue contre le Cancer, e Agence Nationale de la Recherche (Agence Nationale de la Recherche Programa Blanc).

Experimentos cumprir com a regulamentação nacional e comunitária relativa à protecção dos animais utilizados para fins científicos e com princípios internacionalmente consagrados de substituição, redução e aperfeiçoamento. 1. Preparação de Pratos fibronectina revestidos 14 <ol><li> Pipetar 500 ml de 10 mg / ml de fibronectina de grau de cultura diluído em 1x tampão fosfato salino (PBS) para uma placa de Petri de plástico padrão (Ø 40 x 11 mm). Incubar a 37 ° C durante 1 hora. Nota: se estiver usando pratos de vidro ou lamelas de vidro para imagens subsequentes, use a / ml solução fibronectina 100 mg. </li><li> Lavar três vezes com PBS 1x. </li><li> Substituir PBS com 500 ml de 1x PBS/0.1% Albumina de Soro Bovino (BSA). Incubar a 37 ° C durante 30 min. </li><li> Lavar três vezes com PBS 1x. </li><li> Encha-se com PBS 1x e manter a 4 ° C até à sua utilização (geralmente de 24-48 h). </li></ol> 2. Dissecção dos explantes NC &lt;ol&gt; <li> Preparar 3/4 normal anfíbio Médio (NAM) (NaCl 110 mM, KCl 2 mM, 1 mM de Ca (NO3) 2, 1 mM de MgSO4, 0,1 mM de EDTA, 1 mM de NaHCO 3, 0,2 x PBS, 50 mg / mL gentamicina 10,15). Não mantenha mais de 1 semana para dissecação, armazenar a 4 ° C. NAM mais podem ser utilizados para a preparação de pratos revestidos-de dissecação de agarose (abaixo). </li><li> Prepare 60 milímetros placas de Petri para dissecção, revestidos com 10 ml de 2% de agarose em 3/4 NAM. </li><li> Recolhe ferramentas de dissecação: pipetas de Pasteur de plástico, um pino mais fino inseto montado no suporte do pino (ver tabela materiais de referência), uma &quot;faca sobrancelha&quot; (feito com o cabelo da sobrancelha embebidos em parafina, na ponta de uma pipeta de Pasteur de vidro 16), dois fina fórceps de dissecação, pipeta P1000 e dicas, um estereoscópio binocular com ampliação de 8-40X 50, uma fonte de luz brilhante, com guias de fibras ópticas. Todas as ferramentas de dissecação devem ser mantidos limpos, depois de cada experimento, lavar duas vezes com água destilada, uma vez wiª etanol 100% e deixar secar. Guarde longe da poeira. </li><li> Preencha o prato dissecção com NAM fresco quase até o topo. </li><li> Obter X. laevis embriões de acordo com os procedimentos padrão 10 e idade deles até atingirem neurula fase 16 (de acordo com Nieuwkoop e Faber tabela de desenvolvimento 17). </li><li> Coloque fase 16 embriões no prato dissecção usando o Pasteur pipeta de plástico. </li><li> Realizar todas as etapas subseqüentes sob o escopo binocular. Retirar a membrana vitelina, com dois pares de pinças. Tenha cuidado para não danificar a parte dorsal dos embriões. </li><li> Espere até que os embriões atingiram fase 17 para iniciar a dissecção. Fase 17 é adequado uma vez que a crista neural é claramente separado do tubo neural, em contraste com as fases anteriores. Após fase 17, NC começa a migrar e se misturando com o mesênquima circundante. </li><li> Cave um buraco pequeno na agarose com uma pinça para fazer um nicho para os embriões, a fim de manterlos durante a dissecção. Coloque um embrião dorsal voltada para cima. Nota: massa de modelar pode ser utilizada para manter os embriões em vez escavar um buraco no agarose. </li><li> Faça uma incisão na parte lateral da prega neural anterior com a faca sobrancelha eo pino do inseto. Tenha cuidado para não ir muito fundo no embrião: destinam-se a cortar 2-3 camadas celulares (nunca chegam tão profundo como o archenteron!). Durante todo o processo de esvaziamento, nunca permitir que os tecidos dissecados para entrar em contacto com a interface ar-líquido desde X. tecidos embrionários laevis são lisadas pela tensão superficial. </li><li> Faça uma segunda incisão paralela na parte mais dorsal do neural dobra usando os mesmos instrumentos. </li><li> Se necessário, gire o prato em torno de 90 °. Fazer uma terceira incisão perpendicular com a faca sobrancelha e o pino de insectos, no lado posterior dos dois primeiros incisões. O NF é uma massa espessa de células debaixo da camada exterior pigmentada de ectoderma, lateralpara o tubo neural. </li><li> Utilize a ponta da faca sobrancelha para separar a camada pigmentada da ectoderme NC subjacente, a partir da terceira borda da incisão. </li><li> Usar o lado da faca sobrancelha para separar o tecido de NC a partir da terceira incisão. O tecido NC é bastante acinzentada, enquanto a mesoderme cefálica subjacente é branco. Separe cuidadosamente o NC a partir da mesoderme. </li><li> Retire o tecido NC anteriormente da vesícula óptica, e cortar a este nível para liberar o explante. Como um controlo para a primeira experiência, o controlo de expressão snail2 (um marcador NC canónica) em alguns explantes por hibridação in situ é recomendada. </li></ol> 3. Galvanização explantes sobre fibronectina <ol><li> Preencha o prato revestido com fibronectina Modificado Danilchick Médio (MDM, NaCl 53 mM, Na2CO 3 5 mM, K gluconato de 4,5 mM, Na Gluconate 32 mM, MgSO4 1 mM, CaCl2 1 mM, BSA 0,1%, ajuste de pH a 8.3 com 1 M de Bicina <sup&gt; 18). </li><li> Transferir os explantes no prato revestido com fibronectina com uma pipeta P1000. Nunca permitir que os explantes de tocar a interface ar-líquido. Primeiro, um pouco de líquido pipeta na ponta, em seguida, os explantes, em seguida, um pouco de líquido novamente. Não permita que o ar para a ponta durante a transferência dos explantes no prato revestido de fibronectina. Uma vez que a ponta está dentro do líquido do prato revestido de fibronectina, deixe-os explantes lentamente descer por gravidade, ou empurrar gentilmente. Evite ejetar os explantes rápido demais. </li><li> Coloque 10-30 explantes no prato usando a faca sobrancelha. Evitar os lados do prato, que não são adequados para a imagiologia subsequente. </li><li> Tente não mover o prato até os explantes ter anexado a fibronectina (leva cerca de 15-30 minutos). Se necessário, mova o prato com muito cuidado. </li><li> Coloque o prato em uma incubadora de refrigeração (entre 15-20 ° C, para ver gráfico de temperatura 17). Quando colocado a 15 ° C, as células de are geralmente migrando de forma eficiente e dispersão após 6 horas de incubação (este tempo pode variar entre os experimentos). </li></ol> 4. Enxertia explantes em embriões de host <ol><li> Corte uma lamela de vidro fino em pedaços muito pequenos, usando uma pinça grossa. O tamanho das peças deve ser de cerca de 1,5 mm 2. Mergulhar os pedaços num prato de Petri contendo 3/4 de NAM ou PBS utilizando fórceps. </li><li> Preparação do embrião hospedeiro: dissecar o anfitrião NC em 3/4 NAM como descrito em 3) com um cuidado especial para não danificar o embrião hospedeiro durante a remoção da membrana vitelina e dissecando o NC. A tensão do tecido tende a aumentar o tamanho da ablação de: permitir que o embrião hospedeiro curar parcialmente dissecando o embrião dador. </li><li> Em outro prato revestido de agarose ou em outra parte do mesmo prato, dissecar o explante NC a partir do embrião dador, tal como descrito no protocolo 3. Transferir o enxerto NC no prato contendo o hospedeiro com cuidado, como descrito emProtocolo no 3. </li><li> Imediatamente coloque o explante NC para a área de acolhimento ablated usando uma pinça. O explante deve anexar parcialmente. Coloque um pedaço de lamela de vidro para o embrião enxertada para manter o enxerto no lugar. Escolha um pedaço de vidro maior que o embrião para evitar danificá-lo. O embrião vai ser um pouco achatado. </li><li> Aguarde pelo menos 15-20 min, em seguida, remova cuidadosamente a lamela. Deixe o embrião recuperar durante mais 10 minutos e cuidadosamente pipetar-lo em um prato limpo preenchido com 3/4 de NAM, utilizando a pipeta de 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>

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Este protocolo descreve uma técnica fácil de explantar premigratory craniano NC em embriões laevis X.. Os embriões utilizados para tais experimentos precisam ser robusto e curar bem. Descarte qualquer lote de embriões saudáveis. Além disso, os embriões crescem a diferentes temperaturas (de 12 a 18-20 ° C), de modo a excisar crista neural na fase 17, e não depois. Após a fase 17, crista neural pode ser misturado com mesoderme cefálica e não pode ser removido completamente. Tecidos Xenopus c...

A crista neural (NF) é uma população de células embrionárias transiente que surge a partir do tubo neural, no final de neurulação em embriões de vertebrados. Os eventos de sinalização e genéticos que controlam a especificação NC começar tão cedo quanto a gastrulação. O NC é especificado na fronteira entre o ectoderma neural e não neural por sinais de tecidos dorsais circundantes. No final da neurulação, células NC sofrer uma transição epitélio para mesênquima (EMT) e migrar extensivamente no embrião seguindo vias estereotipados por responder ao redor pistas orientadoras. Uma vez que eles chegaram ao seu destino final, eles se diferenciam em uma vasta gama de derivados, por exemplo, neurônios, glia, ossos, cartilagens e células pigmentadas 1-5. Por causa de sua contribuição para muitos tipos de células e tecidos embrionários, defeitos em qualquer etapa do desenvolvimento NC células, a partir de indução de diferenciação final, pode causar síndromes congênitas nomeados neurocristopathies 6. Emanipulação Xperimental do NC desenvolvimento em diferentes estágios - especificação, EMT, migração e diferenciação - irá melhorar a nossa compreensão da neurocristopathies e permitir desenho de estratégias terapêuticas potenciais. O embrião de Xenopus laevis é um modelo de escolha para estudar o desenvolvimento NC. Um grande número de embriões são fáceis de obter, e fecundação externa dá acesso aos primeiros passos do desenvolvimento. Muitas ferramentas estão disponíveis para manipular experimentalmente X. laevis desenvolvimento embrionário. Gene ganho de função e knockdown são fáceis de realizar por microinjecting células individuais dos primeiros blástulas. Tecidos embrionários podem ser cortados para in vitro reassociation 7-11 ou ensaios de back-enxerto 12,13. Neste protocolo, descrevemos como dissecar premigratory NC craniano em X. laevis neurulas final, antes da migração. Estes explantes podem ser cultivados em fibronectina coateplacas d para estudar a migração e diferenciação em controladas em condições experimentais in vitro. Explantes NC também pode ser enxertado em embriões hospedeiros normais ou manipuladas para estudar a sua migração e diferenciação in vivo.

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	<tbody>
		<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

A crista neural (NF) é uma população de células do tubo neural dorsal transiente que sofre uma transição epitélio para mesênquima (EMT) no final de neurulação, migra extensivamente para vários órgãos, e diferencia-se em vários tipos de derivados de (neurónios, células da glia, cartilagem e osso, as células pigmentadas e endócrino). Neste protocolo, descrevemos como dissecar o premigratory craniano NC a partir de embriões de Xenopus laevis, a fim de estudar o desenvolvimento NC in vivo e in vitro. O modelo sapo oferece muitas vantagens para estudar o desenvolvimento precoce; lotes abundantes estão disponíveis, os embriões se desenvolvem rapidamente, no ganho vivo e perda de estratégias de função permitem a manipulação da expressão gênica antes NC dissecção em doador e / ou embriões de acolhimento. Os explantes de NC podem ser semeadas em fibronectina e utilizados para estudos in vitro. Eles podem ser cultivadas durante vários dias, num meio definido isento de soro. Nós também descrevem como enxertar explantes NC voltaem embriões de acolhimento para estudar a migração NC e diferenciação in vivo.

Quando plaqueadas em fibronectina, os explantes da crista neural anexar-se rapidamente (15-30 min) e o explante estende horizontalmente dentro de 2 horas (Figura 1A). Depois de 3-6 células hr começar a se dispersar. Em 24 horas, a 15 ° C, muitas células começaram a migrar a partir do explante (Figuras 1B e 1C). Yolk torna as células muito brilhante em fase de contraste (Figura 1B). Protuberâncias celulares (filopodes e lamellipodes) são claramen...

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 descreve como dissecar crista neural craniana premigratory (NC) de Xenopus laevis neurulas. Estes explantes podem ser semeadas em fibronectina e cultivadas in vitro, ou enxertados em embriões hospedeiros. Esta técnica permite estudar os mecanismos da transição epitélio NC-to-mesênquima, migração e diferenciação.

Dissecção<em&gt; Xenopus laevis</em&gt; Crista neural para<em&gt; In vitro</em&gt; Cultura Explant ou<em&gt; In vivo</em&gt; Transplante

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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Biology

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

13.2K visualizações.  Centre Universitaire.  Este protocolo descreve como dissecar crista neural craniana premigratory (NC) de Xenopus laevis neurulas. Estes explantes podem ser semeadas em fibronectina e cultivadas in vitro, ou enxertados em embriões hospedeiros. Esta técnica permite estudar os mecanismos da transição epitélio NC-to-mesênquima, migração e diferenciação. 

Vídeo: 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.

Dissecção do Organizador e Explantes Pólo animal a partir de embriões Xenopus laevis e Montagem de um ensaio de Adesão Celular

Biologia

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.

Incorporação de plástico e secção de embriões Xenopus laevis

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.

Obtenção de ovos de Xenopus laevis fêmeas

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.

Preparação e fracionamento de Xenopus laevis Egg Extractos

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.

Microinjeção de Oócitos 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.

Determinação tecido Usando o Transplante Cap Animal (ACT) em Ensaio 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 ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Em células vivas, Imagem e Análise Quantitativa de células embrionárias epiteliais em Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

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.

Produção de transgênicos Xenopus laevis Por Restrição Integração Mediada por Enzima e Transplante 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.

Grampo patch e Técnicas de Perfusão para estudar canais iônicos Expresso em Xenopus Oócitos

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.

Transplantes faciais em<em&gt; Xenopus laevis</em&gt; Os embriões

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

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