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

Forfatterne takker Adeline Dolly for billeder af explant på fibronektin, Eric Theveneau for nyttige diskussioner og Animal Facility fra Institut Curie. CM er en post.doc i Region Ile de France (Domaine d&#39;Interet Majeur Stem Pole), Université Paris Sud (Attache Temporaire d&#39;Enseignement et de Recherche), og Agence Nationale de la Recherche. Dette arbejde blev finansieret af Université Paris Sud (Attractivite 2011), Centre National de la Recherche Scientifique (aktion thématique et Incitative sur-programmet), Association pour la Recherche contre le Cancer Grant SFI20101201882, Ligue contre le Cancer, og Agence Nationale de la Recherche (Agence Nationale de la Recherche Programme Blanc).

Eksperimenter overholder nationale og europæiske bestemmelser om beskyttelse af dyr, der anvendes til videnskabelige formål, og med internationalt vedtagne principper om erstatning, begrænsning og forfinelse. 1.. Fremstilling af fibronectincoatede Dishes 14 <ol><li> Afpipetteres 500 ml 10 mg / ml kultur klasse fibronectin fortyndet i 1x phosphatpufret saltvand (PBS) i et standard plast petriskålen (Ø 40 x 11 mm). Der inkuberes ved 37 ° C i 1 time. Bemærk: Hvis du bruger g...

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Denne protokol beskriver en nem teknik eksplanteres premigratory kraniel NC i X. laevis embryoner. De embryoner, der anvendes til sådanne forsøg skal være robust og helbrede godt. Kassér parti af usunde embryoner. Desuden vokser embryoner ved forskellige temperaturer (fra 12 til 18-20 ° C), med henblik på at udskære neuralkam på 17. etape og ikke senere. Efter trin 17 kan neuralkam blandes med cephalic mesoderm og kan ikke fjernes helt. Xenopus væv heler hurtigt og miste deres adhæsion til an...

De neurale våbenskjold (NC) er en forbigående embryonale celle befolkning, der kommer fra neuralrøret i slutningen af ​​neurulation i hvirveldyr embryoner. Signal-og genetiske begivenheder, der styrer NC specifikation starte så tidligt som gastrulation. NC er angivet på grænsen mellem det neurale og ikke-neurale ectoderm af signaler fra de omkringliggende dorsale væv. Ved udgangen af ​​neurulation NC celler gennemgår en epitel-til-mesenchyme overgang (EMT), og i vidt omfang migrere i embryoet efter stereotype ruter ved at svare på omgivende vejledende signaler. Når de har nået deres endelige bestemmelsessted, de differentierer til en bred vifte af derivater fx neuroner, glia, knogler, brusk og pigmenterede celler 1-5. På grund af deres bidrag til mange celletyper og væv fra fostre, fejl på ethvert trin af NC-celler udvikling, fra induktion til den endelige differentiering, kan forårsage medfødte syndromer navngivne neurocristopathies 6. Experimental manipulation af udviklingen NC på forskellige stadier - Specifikation, EMT, migration og differentiering - vil forbedre vores forståelse af neurocristopathies og tillade design af potentielle terapeutiske strategier. Xenopus laevis embryo er en model af valg for at studere NC udvikling. Et stort antal embryoner er nemme at få, og ekstern befrugtning giver adgang til de allerførste skridt i udviklingen. Mange værktøjer er tilgængelige for eksperimentelt manipulere X. laevis fosterudviklingen. Gene gain-of-funktion og knockdown er nemme at udføre ved mikroinjektion individuelle celler af tidlig blastulas. Embryonale væv kan skæres til in vitro reassociation 7-11 eller back-podning assays 12,13. I denne protokol, beskriver vi, hvordan at dissekere ud premigratory kranie NC i X. laevis sene neurulas forud for migration. Disse eksplantater kan dyrkes på fibronectin-Coated plader at studere migration og differentiering i kontrolleret in vitro eksperimentelle betingelser. NC explanter kan også blive podet ind i normale eller manipulerede værten embryoner til at studere deres migrering og differentiering 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>
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		<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>
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		<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>
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dissection of xenopus laevis neural crest for in vitro explant culture or in vivo transplantation

De neurale våbenskjold (NC) er en forbigående dorsal neuralrøret cellepopulation, som gennemgår et epithel-til-mesenchyme overgang (EMT) i slutningen af ​​neurulation, vandrer udstrakt i retning af forskellige organer, og skelner i mange typer af derivater (neuroner, glia, brusk og knogler, pigmenterede og endokrine celler). I denne protokol, beskriver vi, hvordan at dissekere premigratory kranie NC fra Xenopus laevis embryoner, for at studere NC udvikling in vivo og in vitro. Frøen model giver mange fordele til at studere tidlige udvikling, rigelige portioner er tilgængelige, embryoner udvikler sig hurtigt, in vivo gevinst og tab af funktion strategier giver manipulation af genekspression før NC dissektion i donor-og / eller værten embryoner. NC eksplantaterne kan være belagt på fibronektin og anvendes til in vitro-undersøgelser. De kan dyrkes i adskillige dage i et serum-frit defineret medium. Vi beskriver også hvordan man pode NC eksplanteret tilbagei host embryoner til at studere NC migration og differentiering in vivo.

Når belagt på fibronektin, de neurale crest eksplantaterne vedhæfte hurtigt (15-30 min) og eksplantatet spreder flad inden 2 timer (Figur 1A). Efter 3-6 timers celler begynder at sprede. Ved 24 timer ved 15 ° C, har mange celler er begyndt at vandre væk fra eksplantatet (Fig. 1B og 1C). Æggeblomme gør cellerne meget lyse under fasekontrast (figur 1B). Cell fremspring (filopodes og lamellipodes) er tydeligt synlige efter phalloidin farvning af akt...

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Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation (Video) | JoVE

Denne protokol beskriver, hvordan man dissekere premigratory kraniel neuralkam (NC) fra Xenopus laevis neurulas. Disse eksplantater kan udpladet på fibronectin og dyrket in vitro, eller podes tilbage i værten embryoner. Denne teknik gør det muligt at studere de mekanismer NC epitel-til-mesenchyme overgang, migration og differentiering.

Dissektion af<em&gt; Xenopus laevis</em&gt; Neural Crest<em&gt; In vitro</em&gt; Eksplantat kultur eller<em&gt; In vivo</em&gt; Transplantation

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

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay (Video) | JoVE

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic Embedding and Sectioning of Xenopus laevis Embryos (Video) | JoVE

Obtaining Eggs from Xenopus laevis Females

Obtaining Eggs from Xenopus laevis Females (Video) | JoVE

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

Preparation and Fractionation of Xenopus laevis Egg Extracts (Video) | JoVE

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 (Video) | JoVE

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 (Video) | JoVE

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

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis (Video) | JoVE

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

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation (Video) | JoVE

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 (Video) | JoVE

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 (Video) | JoVE

Facial Transplantation i<em&gt; Xenopus laevis</em&gt; Embryoner

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