Name: in ovo electroporation of mirna-based plasmids in the developing neural tube and assessment of phenotypes by dii injection in open-book preparations
Uploaded: 2012-10-16T12:00:00
Description: Watch this Scientific Journal Video about In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations at JoVE.c

Werken in het lab van ES wordt ondersteund door de Zwitserse National Science Foundation. We willen graag Dr Beat Kunz bedanken voor hulp bij het filmen.

1. Bereiding van RNAi plasmide-DNA voor celtype-specifieke gen-silencing Plasmiden (figuur 1) gesynthetiseerd met behulp van standaard moleculaire kloneringstechnieken, zoals eerder beschreven 3,4. 1,1 Klonen in de vectoren: oligonucelotide ontwerp <ol><li> We gebruiken dezelfde universele oligonucleotiden en het klonen van protocollen die worden beschreven in de productinformatie die bij de pRFPRNAiC vector 4 (ARK-Genomics)....

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Deze eenvoudige vector-gebaseerde kunstmatige miRNA expressie strategie kan worden gebruikt om knockdown endogene genexpressie in de kip neurale buis. Deze functionele tools bieden meerdere gene silencing, tijdelijke controle en cel-type specificiteit, de interpretatie van complexe ontwikkelingstrajecten te vergemakkelijken. In het bijzonder hebben we aangetoond dat de bruikbaarheid van deze plasmiden in commissurale axon leiding, aangezien de plasmiden kunnen worden gebruikt om verschillende genen knockdown in commissu...

<table border="1"><tbody><tr><td> Naam van het reagens </td><td> Vennootschap </td><td> Catalogusnummer </td></tr><tr><td> 0,5 mm glazen capillairen </td><td> Wereld Precisie-instrumenten </td><td> 1B120F-4 </td></tr><tr><td> Glas naald trekker </td><td> Narishige </td><td> PC-10 </td></tr><tr><td> Elektroporator </td><td> BTX </td><td> ECM 830 </td></tr><tr><td> Sylgard siliconenelastomeer </td><td> Wereld Precisie-instrumenten </td><td> SYLG184 </td></tr><tr><td> Wolfraamdraad, 0,075 mm </td><td> Wereld Precisie-instrumenten </td><td> TGW0325 </td></tr><tr><td> Insect pennen, 0,20 mm </td><td> Fine Science Tools </td><td> 26.002-20 </td></tr><tr><td> Insect pennen, 0,10 mm </td><td> Fine Science Tools </td><td> 26.002-10 </td></tr><tr><td> Lente schaar </td><td> Fine Science Omolen </td><td> 15.003-08 </td></tr><tr><td> Dumont # 5 forceps </td><td> Fine Science Tools </td><td> 11.252-20 </td></tr><tr><td> Dumont # 55 tang </td><td> Fine Science Tools </td><td> 11.255-20 </td></tr><tr><td> Snel Dil </td><td> Molecular Probes </td><td> D-7756 </td></tr><tr><td> TL-microscopen </td><td> Olympus </td><td> SZX12, BX51 </td></tr></tbody></table>

in ovo electroporation of mirna-based plasmids in the developing neural tube and assessment of phenotypes by dii injection in open-book preparations

Commissurale DI1 neuronen zijn uitgebreid bestudeerd om de mechanismen axon begeleiding bij ontwikkeling 1,2 helderen. Deze neuronen in de dorsale ruggenmerg en sturen hun axonen langs stereotype trajecten. Commissurale axonen in eerste instantie ventraal projecteren richting en dan over de vloerplaat. Na het oversteken van de middellijn, deze axonen maken een scherpe bocht rostrale en project lengterichting naar de hersenen. Elk van deze stappen wordt geregeld door de gecoördineerde activiteiten van aantrekkende en afstotende begeleiding cues. De juiste interpretatie van deze signalen is cruciaal voor de begeleiding van axonen langs hun afgebakende pad. Aldus wordt de fysiologische bijdrage van een bepaalde molecule te commissurale axon begeleiding voorkeur onderzocht in de context van de levende embryo. Bijgevolg moet gen knockdown in vivo nauwkeurig worden gecontroleerd om zorgvuldig onderscheid te maken axon begeleiding activiteiten van genen die kunnen spelen meerdererollen tijdens de ontwikkeling. Hier beschrijven we een methode om knockdown genexpressie in de kip neurale buis op een celtype-specifieke, traceerbare wijze. We gebruiken novel plasmidevectoren 3 herbergen celtype-specifieke promoters / enhancers die de expressie van een fluorescerend eiwit marker rijden, direct gevolgd door een miR30-RNAi transcript 4 (binnen de 3&#39;-UTR van het cDNA dat codeert voor het fluorescent protein) ( figuur 1). Wanneer geëlektroporeerd in het ontwikkelende neurale buis, deze vectoren efficiënt opwekken downregulatie van genexpressie en expressie helder fluorescente merker eiwitten kunnen direct traceren van de cellen ervaren knockdown 3. Het mengen van verschillende RNAi vectoren voor elektroporatie kan gelijktijdig knockdown van twee of meer genen in onafhankelijke delen van het ruggenmerg. Dit maakt complexe cellulaire en moleculaire interacties worden onderzocht tijdens de ontwikkeling, op een manier die snel, suitvoering, nauwkeurig en goedkoop. In combinatie met Dil tracing van commissurale axon trajecten in open-boek voorbereidingen 5, deze methode is een handig hulpmiddel voor in vivo studies van de cellulaire en moleculaire mechanismen van commissurale axongroei en begeleiding. In principe kan elke promoter / enhancer worden gebruikt, mogelijk waardoor de techniek breder toepasbaar voor in vivo studies van genfunctie tijdens de ontwikkeling 6. Deze video toont eerst hoe naar en venster eieren, de injectie van DNA plasmiden in de neurale buis en de elektroporatie procedure. Om commissurale axon begeleiding te onderzoeken, wordt het ruggenmerg verwijderd van het embryo als een open boek voorbereiding, vast, en geïnjecteerd met Dil zodat axon paden te traceren. Het ruggenmerg wordt tussen dekglaasjes en gevisualiseerd met behulp van confocale microscopie.

Watch this Scientific Journal Video about In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations at JoVE.c

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

Een methode waarmee genexpressie in de neurale buis downgereguleerd in een celtype-specifieke, traceerbare wijze beschreven. We laten zien hoe<em&gt; In-ovo</em&gt; Elektroporatie van microRNA gebaseerde plasmiden spatiotemporeel gecontroleerde RNA interferentie opwekken kan worden gebruikt om commissurale axon begeleiding onderzoeken in ontwikkelingslanden neurale buis.

In ovo Elektroporatie van miRNA gebaseerde Plasmiden in de ontwikkelingslanden neurale buis en Beoordeling van de fenotypes van Dil Injectie in de Open-boek Voorbereidingen

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are ...

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In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

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