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

Arbejdet i laboratoriet af ES er støttet af den schweiziske National Science Foundation. Vi vil gerne takke Dr. beat Kunz for at få hjælp med at filme.

1. Fremstilling af RNAi Plasmid-DNA til Celletype-specifik gene silencing Plasmider (figur 1) syntetiseres under anvendelse af standard molekylære kloningsteknikker, som tidligere beskrevet i detaljer 3,4. 1,1 kloning i vektorer: oligonucelotide design <ol><li> Vi bruger de samme universelle oligonukleotider og kloning protokoller, der er beskrevet i produktinformationen, der følger med pRFPRNAiC vektor 4 (ARK-Genomics). <o...

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Denne enkle, vektorbaseret kunstige miRNA ekspression strategi kan anvendes til at knockdown endogen genekspression i kylling neuralrøret. Disse funktionelle værktøjer har multiple gen-nedregulering, tidsmæssig regulering og celletype specificitet, for at lette opklaringen af ​​komplekse udviklingsmæssige forløb. Især har vi demonstreret anvendeligheden af disse plasmider i kommissurale Axon vejledning, eftersom plasmider kan anvendes til at knockdown forskellige gener i kommissurale neuroner eller i deres me...

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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

Kommissurale DI1 neuroner er blevet grundigt undersøgt for at belyse de mekanismer, der ligger til grund Axon vejledning under udvikling 1,2. Disse neuroner er beliggende i den dorsale rygmarv og sende deres axoner langs stereotype baner. Kommissurale axoner i første omgang rage ventralt mod og derefter på tværs af gulvpladen. Efter at have krydset midterlinjen gør disse axoner en skarp rostral sving og projekt på langs mod hjernen. Hvert af disse trin er reguleret af koordinerede aktiviteter tiltrækkende og frastødende vejledning cues. Den korrekte fortolkning af disse signaler er afgørende for vejledning af axoner langs deres afgrænsede bane. Således er den fysiologiske bidrag et givet molekyle til kommissurale axon vejledning ideelt undersøgt i forbindelse med levende embryo. Derfor skal gen knockdown in vivo styres præcist med henblik på at skelne omhyggeligt axon vejledningsaktiviteter af gener, der kan spille flereroller under udvikling. Her beskriver vi en fremgangsmåde til knockdown genekspression i kyllingen neuralrøret i en celletype-specifik, sporbar måde. Vi anvender hidtil ukendte plasmidvektorer 3 huser celletype-specifikke promotorer / enhancere der driver ekspressionen af en fluorescerende protein-markør, efterfulgt direkte af en miR30-RNAi transcript 4 (placeret i 3&#39;-UTR af det cDNA, der koder for det fluorescerende protein) ( figur 1). Når elektroporeret i det udviklende neuralrør, disse vektorer fremkalde effektive nedregulering af genekspression og udtrykke stærkt fluorescerende markørproteiner at muliggøre direkte sporing af cellerne oplever knockdown 3. Blande forskellige RNAi vektorer før elektroporering tillader samtidig knockdown af to eller flere gener i uafhængige regioner af rygmarven. Dette tillader komplekse cellulære og molekylære vekselvirkninger, der skal undersøges under udviklingen, på en måde, der er hurtig, sgen, præcis og billig. I kombination med Dil opsporing af kommissurale axon baner i open-book præparater 5, er denne metode et nyttigt værktøj for in vivo studier af de cellulære og molekylære mekanismer af kommissurale Axon vækst og vejledning. I princippet kan enhver promotor / enhancer kan anvendes, potentielt gør teknikken mere bredt anvendelig til in vivo-undersøgelser af genfunktion under udvikling 6. Denne video først viser, hvordan man håndterer og vinduer æg, injektion af DNA plasmider i neuralrøret og elektroporation procedure. At undersøge kommissurale Axon vejledning, er rygmarven fjernet fra embryoet som en åben-book præparat, fikseret, og injiceret med Dil så axon veje kan spores. Rygmarven er monteret mellem dækglas og visualiseret ved hjælp af konfokal mikroskopi.

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

En fremgangsmåde, hvorved genekspression i neuralrøret kan være nedreguleret i en celletype-specifik, sporbar måde beskrives. Vi viser, hvordan<em&gt; In ovo</em&gt; Elektroporation af microRNA-baserede plasmider, som fremkalder spatiotemporally styret RNA-interferens kan anvendes til at undersøge kommissurale Axon vejledning i den udviklende neuralrør.

I ovo Elektroporation af miRNA-baserede plasmider i udviklingslandene neuralrøret og vurdering af fænotyper ved Dil Injection i Open-bog Forberedelser

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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