Name: in ovo expression of microrna in ventral chick midbrain
Uploaded: 2013-09-16T07:00:00
Description: Watch this Scientific Journal Video about In ovo Expression of MicroRNA in Ventral Chick Midbrain at JoVE.com

Vi anerkender K. Mikic, der har bidraget til den indledende fase af denne film og M. Nicolescu for miR billedet. C. Huber blev støttet af et fællesskab af IZKF af Universtitätsklinikum Tübingen, A. Alwin Prem Anand af Fortune program Universtitätsklinikum Tübingen.

1.. Krav til In ovo Elektroporation <ol><li> Forberedelse af Vektorkonstruktionen: miR sense-og antisense-primere blev udformet efter instrukser fra Ambion, udglødet og ligeret i Apal / EcoRI fordøjet pSilencer U6.1 vektor. Efter vellykket transformation en af ​​de resulterende kloner blev dyrket og pSilencer plasmid blev isoleret ved basisk lysis metoden i en Quiagen midi forberedelse kit. </li><li> Elektroder: Elektroder kan købes eller nemt blive bygget 13.</su...

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Denne video viser en effektiv metode til at transficere plasmid ind i de neuroepitelceller for specifikke områder af chick midthjernen. Rektangulære elektriske impulser med lav spænding kan indføre DNA i celler af chick neuralrøret in ovo 6,16. Imidlertid er nøjagtigheden af ​​DNA målretning ofte hindret af den brede elektriske felt, som stiger op gennem de relativt store elektroder (Φ = 0,5 mm). Vi forsøgte at løse dette problem ved hjælp af elektroder mindre i diameter følge retning...

Anerkendelsen af ​​små ikke-kodende RNA som ekstra spillere til genekspression lanceret en ny kompleksitet til genomisk programmering / genregulering. Forskellige arter af ikke-kodende RNA har funktionel betydning i neurale celler, herunder små ikke-kodende RNA 1-4. MicroRNA (MIR eller miRNA) for eksempel viser forskellige og skiftende udtryk profiler i udviklingslandene hjerner 5. Målrettet in ovo elektroporation af kyllingeembryoer giver en enestående mulighed for tidslige og rumlige kontrol af genekspression og lyddæmpning under udviklingen. Denne video viser de forskellige trin i at udføre ektopisk udtryk for Mirs i bestemte områder af chick midthjernen hjælp i ovo elektroporation 6-10. For at sikre en langvarig effekt af disse små ikke-kodende RNA i cellerne, blev DNA-sekvensen af ​​Mirs klonet i mono-eller bi-cistroniske vektorer. For i ovo elektroporation, miR indeholdende vektor injiceres i midthjernen neuralrøret ved at udsætte embryo efter at et lille vindue i æggeskallen. Til transfektion af specifikke områder af midthjernen lille plus (anode) og minus (katode) Platin elektroder placeres på bestemte positioner. For ventrale midthjernen transfektion, er anoden placeret under den venstre ventrale midthjernen og katoden over den højre halvdel af midthjernen, før påføring af en strøm. Åbningen i æggeskallen er lukket med tape og embryoner inkuberes i så længe som nødvendigt for enhver analyse. Denne metode blev oprindeligt beskrevet af Muramatsu et al. 6 og forbedret af Momose et al. 8 for specifikt område transfektion. <img src="/files/ftp_upload/50024/50024schematic1.jpg" fo:content-width-="6in" fo:src="/files/ftp_upload/50024/50024schematic1highres.jpg"/> Skematisk oversigt. <ol><li> Embryoet i ægget er eksponeret ved at skære et lille vindue i the æggeskal. </li><li> Det opløste vektor (er) injiceres i midthjernen under anvendelse af en mikrokapillarbøsninger. </li><li> To elektroder - placeret parallelt eller over og under embryo - generere et pulserende elektrisk felt. </li><li> Det elektriske felt tidsmæssigt skaber porer i cellemembranen, der letter indgang i cellen ved den negativt ladede DNA (eller RNA), tiltrækkes til anoden 11,12. </li></ol>

<table border="1">
<tbody>
 <tr>
 <td>Name </td>
 <td>Company</td>
 <td></td>
 <td>Model</td>
 </tr>
 <tr>
 <td>Borosillicate glass capillaries </td>
 <td>Hartenstein</td>
 <td></td>
 <td>Model: 0.9 mm</td>
 </tr>
 <tr>
 <td>Microcapillary puller</td>
 <td>WPI, Berlin</td>
 <td></td>
 <td>Model: Pul1-E</td>
 </tr>
 <tr>
 <td>Electroporator</td>
 <td>Intracel</td>
 <td></td>
 <td>Model: TSSIC</td>
 </tr>
 <tr>
 <td>Stereomicroscope - fluorescence </td>
 <td>LEICA</td>
 <td></td>
 <td>Model: MZFLIII</td>
 </tr>
 <tr>
 <td>Stereomicroscope</td>
 <td>Zeiss</td>
 <td></td>
 <td>Model: Stemi</td>
 </tr>
 <tr>
 <td>Camera and software</td>
 <td>Zeiss</td>
 <td></td>
 <td>Model: Axiocam MRc/ Axiovision Re. 4.8</td>
 </tr>
 </tbody>
</table>

in ovo expression of microrna in ventral chick midbrain

Ikke-kodende RNA er yderligere spillere i regulering af genekspression. Målrettet in ovo elektroporation specifikke områder giver et unikt værktøj til rumlige og tidsmæssige styring af ektopisk microRNA udtryk. Men er temmelig svært at nå for eventuelle manipulationer ventrale hjernens strukturer som ventrale midthjernen. Her viser vi en effektiv måde at elektroporere miRNA i ventrale midthjernen hjælp af tynde platinelektroder. Denne metode giver en pålidelig måde til at transficere bestemte områder af midthjernen og et nyttigt redskab til in vivo-undersøgelser.

Omfanget af midthjernen område transficeret med miR er synlig gennem ekspressionen af GFP fra en reportervektor injiceres sammen med miR udtrykker vektoren (figur 2). Brug af platinelektroder med en diameter på 0,5 mm, og placeres parallelt med AP akse midthjernen fører normalt til transfektion af et bredt område langs DV-og AP-aksen, herunder baghjerne, midthjernen og undertiden diencephalon (figur 2A og B). Størrelsen af ​​elektroderne resultater i en lang elektrisk felt anve...

Watch this Scientific Journal Video about In ovo Expression of MicroRNA in Ventral Chick Midbrain at JoVE.com

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ektopisk udtryk er en teknik til at belyse microRNA rolle i hjernens udvikling. Men rettet mod specifikke områder ved hjælp af in ovo elektroporation er udfordrende. Her viser vi en effektiv måde til selektivt elektroporere ventrale og dorsale midthjernen regioner.

I ovo Angivelse af MicroRNA i Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

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Neuroscience

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

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