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

Arbeid i laboratoriet av ES er støttet av den sveitsiske National Science Foundation. Vi ønsker å takke dr. Beat Kunz for å få hjelp med filming.

1. Utarbeidelse av RNAi Plasmid DNA for Cell typespesifikke Gene Slå av lyd Plasmider (Figur 1) er syntetisert ved hjelp av standard molekylære kloning teknikker, som tidligere beskrevet i detalj 3,4. 1.1 Kloning inn i vektorer: oligonucelotide utforming <ol><li> Vi bruker de samme universelle oligonukleotider og kloning protokoller som er beskrevet i produktinformasjonen som følger med pRFPRNAiC vektor 4 (ARK-Genomics). <...

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Denne enkle, vektorbasert kunstig miRNA uttrykk strategien kan brukes til knockdown endogene genekspresjon i kylling nevralrøret. Disse funksjonelle verktøy tilbyr flere genet stanse, temporal kontroll og celle-type spesifisitet, for å lette klarlegging av komplekse utviklingsforstyrrelser veier. Spesielt har vi demonstrert nytten av disse plasmider i fugedannende axon veiledning, siden plasmidene kan brukes til å knockdown distinkte gener i fugedannende neuroner eller i deres mellomliggende mål, gulvplaten 3....

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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

Fugedannende DI1 nevroner har blitt grundig studert for å belyse mekanismene bak axon veiledning under utvikling 1,2. Disse nevronene ligger i dorsal ryggmargen og sender sine axoner langs stereotype baner. Fugedannende axons utgangspunktet projisere ventrally mot og deretter over gulvplaten. Etter å ha krysset midtlinjen, disse axons gjøre en skarp rostral sving og prosjekt lengderetningen mot hjernen. Hvert av disse trinnene er regulert av koordinerte aktiviteter attraktive og frastøtende veiledning signaler. Den riktige tolkningen av disse stikkordene er avgjørende for veiledning av axoner langs deres avgrensede veien. Således er den fysiologiske bidraget av en bestemt molekyl å fugedannende axon veiledning ideell undersøkt i sammenheng med den levende embryo. Følgelig må genet knockdown in vivo kontrolleres nøyaktig for å nøye skille axon veiledning aktiviteter av gener som kan spille flereroller under utvikling. Her beskriver vi en metode for å knockdown genuttrykk i kylling nevralrøret i en celle type-spesifikke, sporbar måte. Vi bruker romanen plasmidvektorer 3 harboring celletype-spesifikke promotorer / Forsterker som driver ekspresjon av en fluorescerende protein markør, fulgt direkte av en miR30-RNAi transkript 4 (som befinner seg innenfor 3&#39;-UTR av cDNA som koder for den fluorescerende protein) ( Figur 1). Når electroporated i utvikling nevralrøret, disse vektorer lokke fram effektiv nedregulering av genekspresjon og uttrykke lyse fluorescerende markørproteiner å muliggjøre direkte sporing av cellene opplever knockdown 3. Blande ulike RNAi vektorer før electroporation gjør samtidig knockdown av to eller flere gener i selvstendige regioner i ryggmargen. Dette tillater komplekse cellulære og molekylære interaksjoner som skal undersøkes i løpet av utviklingen, på en måte som er rask, sgjennomført, presis og billig. I kombinasjon med Dii tracing av fugedannende axon baner i åpen bok forberedelser 5, er denne metoden et nyttig verktøy for in vivo studier av cellulære og molekylære mekanismer for fugedannende axon vekst og veiledning. I prinsippet kan hvilken som helst promotor / enhancer brukes, potensielt gjør teknikken mer allment anvendelig for in vivo studier av gen-funksjon under utvikling 6. Denne videoen viser først hvordan å håndtere og vindu egg, kan bruk av DNA plasmider inn nevralrøret og electroporation prosedyren. For å undersøke fugedannende axon veiledning, er ryggmargen fjernes fra embryoet som en åpen bok forberedelse, fast, og injisert med Dii å aktivere axon veier som skal spores. Ryggmargen er montert mellom dekkglass og visualisert ved hjelp konfokalmikroskopi.

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 metode som genekspresjon i nevralrøret kan være downregulated i en celletype-spesifikk, sporbar måte er beskrevet. Vi viser hvordan<em&gt; I ovo</em&gt; Electroporation av mikroRNA-baserte plasmider som framprovosere spatiotemporally kontrollert RNA-interferens kan brukes til å undersøke fugedannende axon veiledning i utviklingsland nevralrøret.

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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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