Name: dissection of xenopus laevis neural crest for in vitro explant culture or in vivo transplantation
Uploaded: 2014-03-04T17:00:00
Description: Watch this Scientific Journal Video about Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation at JoVE.com

Yazarlar yararlı tartışmalar ve Institut Curie&#39;nin Hayvan Tesisi için fibronektin, Eric Theveneau üzerinde eksplant resimler için Adeline Dolly teşekkür ederim. CM Bölge Ile de France (Domaine d&#39;Interet Majeur Pole Stem), Universite Paris Sud (Ataşesi TEMPORAIRE d&#39;Enseignement et de Recherche) ve Agence Nationale de la Recherche bir doktora sonrası adam. Bu çalışma Universite Paris Sud (Attractivite 2011), Centre National de la Recherche Scientifique (Aksiyon thématique ve teĢvik sur Programı), Dernek dökün la Recherche contre le Kanser Grant SFI20101201882, Ligue contre le Kanser ve Agence Nationale de la tarafından finanse edildi Recherche (Agence Nationale de la Recherche Programı Blanc).

Deneyler bilimsel amaçlı ve değiştirme, azaltma ve arıtma uluslararası yerleşik ilkeleri ile kullanılan hayvanların korunmasına ilişkin ulusal ve Avrupa yönetmeliğe uygun. 1.. Fibronektin kaplı kaplar 14 hazırlanması <ol><li> Standart bir plastik Petri tabağı (çap: 40 x 11 mm) içine 1x Fosfat Tamponlu Salin (PBS) içinde sulandırılmış 10 mg / ml kültürü dereceli fibronektin Pipet 500 ml. 1 saat boyunca 37 ° C&#39;de inkübe edilir. Not: sonraki görü...

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Bu protokol X. laevis embriyoların premigratory kranial NC eksplantasyona kolay bir teknik anlatılmaktadır. Bu tür deneyler için kullanılan embriyo sağlam olması ve iyileştirmek gerekir. Sağlıksız embriyoların herhangi bir toplu atın. Buna ek olarak, ve daha sonraki bir aşamada 17 nöral tepe tüketim için, (12 ile 18-20 ° C) çeşitli sıcaklıklarda embriyolar büyür. 17 sahne sonra, nöral krest sefalik mezoderm ile karıştırılabilir ve tamamen kaldırılamaz. Ksenopus dokuları...

Sinir ucu (NC), embriyonun içinde nörülasyon sonunda nöral tüpünden çıkan geçici bir embriyonik hücre popülasyonu. NC şartname kontrol sinyalizasyon ve genetik olaylar gibi erken gastrulasyon olarak başlar. NC çevreleyen dorsal dokulardan gelen sinyaller tarafından nöral ve nöral olmayan ektoderm arasındaki sınırda belirtildi. Nörülasyon sonunda, Kuzey hücreleri epitel-to-mezenşimin geçiş (EMT) geçmesi ve yol gösterici ipuçları çevreleyen yanıt vererek kalıplaşmış yolları takip embriyo yoğun göç. Onların nihai hedefe ulaştığında, onlar türevleri, örneğin nöronlar, glia, kemik, kıkırdak, ve pigmentli hücrelerin 1-5 geniş bir diziye ayırt. Çünkü birçok hücre tipleri ve embriyonik dokular, NC hücreleri gelişme herhangi bir aşamasında kusurları, indüksiyon nihai farklılaşma katkıları, neurocristopathies 6 adlı konjenital sendromlara neden olabilir. Dşartname, EMT, göç, ve farklılaşma - - farklı aşamalarında gelişen NC Xperimental manipülasyon neurocristopathies anlayışımızı geliştirmek ve potansiyel tedavi stratejileri tasarımı sağlayacaktır. Xenopus laevis embriyo NC gelişimini incelemek için tercih edilen bir modeldir. Embriyoların çok sayıda elde etmek kolay, ve dış döllenme gelişme ilk adımlardan erişim sağlar. Birçok araç deneysel X işlemek için kullanılabilir embriyo gelişimini laevis. Kazanç fonksiyon-ve demonte gen erken blastulas bireysel hücrelerin microinjecting gerçekleştirmek kolaydır. Embriyonik dokular in vitro tekrar birleşme 7-11 veya sırt-aşılama deneyleri 12,13 için kesilebilir. Bu protokol, biz X. premigratory kranial NC teşrih nasıl açıklar önce göç geç neurulas, laevis&#39;den. Bu eksplantlar, fibronektin boru hattlar üzerinde kültürlenebiliriçinde göç ve farklılaşma çalışma d plakalar vitro deney koşullarında kontrol edilir. NC eksplantlan in vivo olarak göç ve farklılaşma çalışma normal veya manipüle ana embriyolara aşılanabilirler.

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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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</table>

dissection of xenopus laevis neural crest for in vitro explant culture or in vivo transplantation

Nöral krest (NC) nörülasyon sonunda epitel-to-mezenşimin geçiş (EMT) uğrar geçici bir dorsal nöral tüp hücre nüfusu, çeşitli organlara doğru yoğun göç ve türevlerinin birçok türleri (nöronlar, glia içine ayırır kıkırdak ve kemik, pigmentli ve endokrin hücreler). Bu protokol, in vivo ve in vitro NC gelişimini incelemek amacıyla, Xenopus laevis embriyolardan premigratory kafatası NC incelemek nasıl açıklar. Kurbağa modeli, erken gelişimini incelemek için birçok avantaj sunuyor; bol partiler embriyolar in vivo kazanç ve fonksiyon stratejileri kaybı öncesinde donör ve / veya ana embriyoların NC diseksiyon gen ifadesinin değiştirilmesine izin verir, hızla geliştirmek mevcuttur. NC Eksplantlar fibronektin üzerine kaplanmıştır ve in vitro çalışmalar için de kullanılabilir. Bir serumsuz ortam içinde tanımlanmış birkaç gün boyunca kültürlenebilir. Biz de Kuzey Carolina eksplantlarının geri greft nasıl açıklarin vivo NC göç ve farklılaşma çalışmak için ana embriyolara.

Fibronektin üzerinde kaplama yaparken, nöral krest eksplantlar hızla (15-30 dakika) takın ve eksplant 2 saat (Şekil 1A) dahilinde düz yayılır. 3-6 saat hücreler dağılmaya başlar sonra. 15 ° C&#39;de 24 saat sonra, hücreler, bir çok uzak eksplant (Şekil 1B ve 1C) göç etmeye başlamıştır. Sarısı faz kontrast (Şekil 1B) altında hücreleri çok parlak yapar. Hücre çıkıntıların (filopodes ve lamellipodes) aktin hücre iskeletin...

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

Bu protokol Xenopus laevis neurulas gelen premigratory kranial sinir kret (NC) teşrih nasıl açıklanır. Bu eksplantlar, fibronektin ve in vitro kültürlenmiş üzerine kaplanır, ya da arka ana embriyolara aşılanabilirler. Bu teknik NC epitel-to-mezenşimin geçiş, göç, ve farklılaşma mekanizmaları eğitim veriyor.

Diseksiyonu<em&gt; Xenopus laevis</em&gt; Sinir Crest için<em&gt; In vitro</em&gt; Eksplantasyon Kültür veya<em&gt; In vivo</em&gt; Transplantasyon

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

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

Organizatör ve Hayvan Kutbu eksplantlar Xenopus laevis Embriyolar ve Hücre Yapışma Testi Meclisi diseksiyonu

Biyoloji

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastik gömülmesi ve Xenopus laevis Embriyolar Kesit

Obtaining Eggs from Xenopus laevis Females

Xenopus laevis Dişiler Yumurta Alınması

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

Xenopus laevis Yumurta hazırlanması ve Fraksiyonu ayıklar.

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

Xenopus laevis Oosit, Mikroenjeksiyon

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

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

Doku belirlenmesi Hayvan Cap Nakli (ACT) Testi 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

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

Transgenik, üretimi Xenopus laevis Kısıtlama Enzim aracılığı ile Entegrasyon ve Nükleer Nakli

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

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

İyon Kanalları incelenmesi için yama Kelepçe ve Perfüzyon Teknikleri cinsinden ifade edilir Xenopus Oosit

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

Facial Transplants in Xenopus laevis Embryos

Yüz Nakli<em&gt; Xenopus laevis</em&gt; Embriyolar

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