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

Författarna tackar Adeline Dolly för bilderna av Explantation på fibronektin, Eric Theveneau för hjälp diskussioner och Animal Facility av Institut Curie. CM är en forskarassistent i regionen Ile de France (Domaine d&#39;Interet Majeur Stem Pole), Université Paris Sud (Attache Temporaire d&#39;Enseignement et de Recherche), och Agence Nationale de la Recherche. Detta arbete har finansierats av Université Paris Sud (Attractivite 2011), Centre National de la Recherche Scientifique (Action Thematique et Incitative sur Programme), Association pour la Recherche contre le Cancer Grant SFI20101201882, Ligue contre le Cancer, och Agence Nationale de la Recherche (Agence Nationale de la Recherche Programme Blanc).

Experiment uppfyller nationella och EU-förordning om skydd av djur som används för vetenskapliga ändamål och med internationella principer för ersättning, begränsning och förfining. 1. Beredning av fibronektinbelagda Rätter 14 <ol><li> Pipettera 500 ml av 10 mg / ml kulturgrad fibronektin utspätt i 1 x fosfatbuffrad saltlösning (PBS) i ett vanligt plastpetriskål (diameter 40 x 11 mm). Inkubera vid 37 ° C under 1 timme. OBS: Om du använder glas rätter eller täckg...

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Detta protokoll beskriver en enkel teknik för att explantera premigratory cranial NC i X. laevis embryon. De embryon som används för sådana experiment måste vara robust och läker bra. Kassera parti ohälsosamma embryon. Dessutom växer embryon vid olika temperaturer (från 12 till 18 till 20 ° C), i syfte att skära ut neurallisten vid steget 17 och inte senare. Efter etapp 17, kan neurallist blandas med KRANIE mesoderm och kan inte tas bort helt och hållet. Xenopus vävnader läker snabbt och ...

Neurallisten (NC) är en övergående embryonal cellpopulation som framträder i neuralröret i slutet av neurulation i ryggradsdjur embryon. Signalering och genetiska händelser som styr NC specifikation börja så tidigt som gastrulation. NC specificeras vid gränsen mellan neurala och icke-neural ektoderm av signaler från omgivande rygg vävnader. Vid slutet av neurulation, NC celler genomgår ett epitel till mesenkym övergång (EMT) och migrera i stor utsträckning i embryot följande stereotypa rutter genom att svara på omgivande vägledande cues. När de har nått sin slutdestination, de differentieras till ett brett spektrum av derivat, t.ex. nervceller, glia, ben, brosk och pigmenterade celler 1-5. På grund av deras bidrag till många celltyper och embryonala vävnader, defekter vid varje steg av NC-celler utveckling, från induktion till slutlig differentiering, kan orsaka medfödda syndrom heter neurocristopathies 6. Experimental manipulation av utvecklings NC i olika skeden - specifikationer, EMT, migration och differentiering - kommer att öka vår förståelse av neurocristopathies och möjliggöra utformning av potentiella terapeutiska strategier. Xenopus laevis embryot är en modell av val att studera NC-utveckling. Ett stort antal embryon är lätt att få, och yttre befruktning ger tillgång till de allra första stegen i utvecklingen. Många verktyg finns tillgängliga för att experimentellt manipulera X. laevis embryonala utvecklingen. Gene vinst-av-funktion och knockdown är lätta att utföra genom microinjecting enskilda celler i tidiga blastulas. Embryonala vävnader kan klippas för in vitro-återförening 7-11 eller back-ympning analyser 12,13. I detta protokoll, beskriver vi hur man dissekera ut premigratory cranial NC i X. laevis sena neurulas, före migreringen. Dessa explantat kan odlas på fibronektin-coated plattor för att studera migration och differentiering i kontrollerad in vitro experimentella förhållanden. NC explants kan även ympas in i normala eller manipulerade värd embryon för att studera deras migration och differentiering in vivo.

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			<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>
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			<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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			<td height="21" style="height:21px;width:244px;">Stainless Steel Insect Pins. Size 000</td>
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			<td height="42" style="height:42px;width:244px;">Dumont #5 forceps 0.05 mm x 0.02 mm</td>
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dissection of xenopus laevis neural crest for in vitro explant culture or in vivo transplantation

Neurallisten (NC) är en övergående rygg neuralrörsdefekter cellpopulation som genomgår en epitel-till-mesenkym övergång (EMT) i slutet av neurulation, vandrar i stor utsträckning mot olika organ, och skiljer i många typer av derivat (nervceller, glia, brosk och ben, pigmenterade och endokrina celler). I detta protokoll, beskriver vi hur man dissekera premigratory cranial NC från Xenopus laevis embryon, för att studera NC utveckling in vivo och in vitro. Grodan modellen erbjuder många fördelar för att studera tidiga utveckling, rikliga satser finns tillgängliga, embryon utvecklas snabbt, in vivo-vinst och förlust av funktionsstrategier tillåter manipulering av genuttryck före NC dissektion i donator och / eller embryon värd. NC explants kan pläterade på fibronektin och som används för in vitro-studier. De kan odlas i flera dagar i ett serumfritt definierat medium. Vi beskriver också hur man ympa NC explants tillbakai värd embryon för att studera NC migration och differentiering in vivo.

När pläterade på fibronektin, neurallisten explantat fäster snabbt (15-30 min) och explantatet sprider platt inom 2 tim (Figur 1A). Efter 3-6 tim celler börjar att sprida. Vid 24 h vid 15 ° C, har många celler började att migrera bort från explantatet (figurerna 1B och 1C). Äggula gör cellerna mycket ljus i fas kontrast (Figur 1B). Cellutskott (filopodes och lamellipodes) syns tydligt efter phalloidin färgning av aktin cytoskelettet ...

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

Detta protokoll beskriver hur man dissekera premigratory cranial neural crest (NC) från Xenopus laevis neurulas. Dessa explants kan pläterade på fibronektin-och odlas in vitro, eller ympas tillbaka in embryon värd. Denna teknik gör det möjligt att studera mekanismerna för NC epitel-till-mesenkym övergång, migration och differentiering.

Dissekering av<em&gt; Xenopus laevis</em&gt; Neural Crest för<em&gt; In vitro</em&gt; Explantation 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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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

Dissekering av Organizer och Animal explants Pole från Xenopus laevis Embryon och församlingen i en cell Adhesion analys

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plast inbäddning och Sektionering av Xenopus laevis embryon

Obtaining Eggs from Xenopus laevis Females

Skaffa Ägg från Xenopus laevis Kvinnor

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

Upprättande och fraktionering av Xenopus laevis Egg Utdrag

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

Mikroinjektion av Xenopus laevis oocyter

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

Tissue Fastställande Använda Transplant Animal Cap (ACT) analys i 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

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

Live-cell imaging och kvantitativ analys av embryonala epitelceller i 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

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

Produktion av Transgena Xenopus laevis Genom begränsning enzymmedierad Integration och 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 ...

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

Patch Clamp och tekniker perfusion för att studera jonkanaler Uttryckt i Xenopus Oocyter

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

Ansikts Transplantationer i<em&gt; Xenopus laevis</em&gt; Embryon

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