Name: analysis of gene function and visualization of cilia-generated fluid flow in kupffer's vesicle
Uploaded: 2013-03-31T14:00:00
Description: Watch this Scientific Journal Video about Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle at JoVE.com

Vi takker Fiona Foley for utmerket lab støtte og sebrafisk omsorg. Dette arbeidet ble støttet en AHA predoctoral fellesskap å GW (11PRE5730027) og NHLBI tilskudd til HJY (R01HL66292) og JDA (R01HL095690).

Oversikt over Stage-spesifikke sebrafisk embryo injeksjoner Antisens Morpholino oligonukleotider (MO), som binder seg til en målrettet mRNA og forstyrre protein uttrykk fra at avskrift, er mye brukt i gene knockdown (tap-av-funksjon) studier i sebrafisk 13,14. Gene Tools, tilbyr LLC MOS som er merket med enten karboksyfluorescens (avgir grønn fluorescens) eller lissamine (avgir rød fluorescens) for å oppdage MO i injisert embryoer med fluorescerende mikroskopi. Ved å injisere MO...

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Bruke scene-spesifikke injeksjoner for å målrette MO til DFC / KV celle avstamning er en god metode for å studere celle-autonomi genfunksjon og unngå mitogen fenotyper forårsaket av global gene knockdown. Imidlertid kan disse injeksjonene være teknisk utfordrende. Injeksjon av MO mellom 256-celle og 1000-celle stadier kan resultere i tre mulige utfall: 1) MO forblir samlet på injeksjonsstedet, 2) Mo diffunderer gjennom eggeplomme og går DFC / KV celler eller 3) Mo diffunderer gjennom eggeplomme og inngår DFC / ...

<table border="1"><tbody><tr><td> Navn på reagens / Material </td><td> Selskapet </td><td> Katalognummer </td></tr><tr><td> Standard Kontroll oligo-Lissamine merket </td><td> Gene Tools, LLC </td><td></td></tr><tr><td> Custom Rock2b morfolino oligo </td><td> Gene Tools, LLC </td><td></td></tr><tr><td> Fluoresbrite Multifluorescent 0,5 mikron Mikrokuler </td><td> Polysciences, Inc. </td><td> 24054 </td></tr></tbody></table>

analysis of gene function and visualization of cilia-generated fluid flow in kupffer's vesicle

Indre organer som hjerte, hjerne, og gut utvikle venstre-høyre (LR) asymmetrier som er kritiske for sine normale funksjoner en. Bevegelige flimmerhårene er involvert i å etablere LR asymmetri i virveldyr embryoer, inkludert mus, frosk, og sebrafisk 2-6. Disse &quot;LR cilia &#39;generere asymmetrisk væskestrømmen som er nødvendig for å utløse en konservert asymmetrisk Nodal (TGF-β superfamily) signaliserer cascade i venstre lateral plate mesoderm, som er tenkt å gi LR mønster for utformingen organer 7. Derfor, for å forstå mekanismene bak LR mønster, er det viktig å identifisere gener som regulerer organiseringen av LR ciliated celler, motilitet og lengden LR cilia og deres evne til å generere robuste asymmetrisk flyt. I sebrafisk embryo, er LR flimmerhårene ligger i Kupffer sin vesikkel (KV) 2,4,5. KV består av et enkelt lag av monociliated epitelcellersom omslutter en væskefylt hulrom. Fate kartlegging har vist at KV er avledet fra en gruppe av ~ 20-30 celler kjent som dorsal forløperen celler (DFCS) som vandrer på dorsal blastoderm marginen under epiboly stadier 8,9. Under tidlig somitt stadier, til DFCS klynge og skille ut ciliated epitelceller danne KV i tailbud av embryoet 10,11. Evnen til å identifisere og spore DFCS-i kombinasjon med optisk åpenhet og rask utvikling av sebrafisk embryo-make sebrafisk KV en utmerket modellsystem for å studere LR ciliated celler. Interessant, stamfedre av DFC / KV celle avstamning beholde cytoplasmatiske broer mellom eggeplomme celle opp til 4 timers post-fertilisering (HPF), mens cytoplasmatiske broer mellom eggeplomme celle og andre embryonale celler tett etter to HPF 8. Dra nytte av disse cytoplasmatiske broer, har vi utviklet en scene-spesifikk injeksjon strategi om å levere morfolino oligonucleotides (MO) utelukkende DFCS og knockdown funksjonen til en målrettet genet i disse cellene 12. Denne teknikken skaper kimære embryoer som gen-funksjon er slått ned i DFC / KV avstamning utvikling i sammenheng med en vill-type embryo. Å analysere asymmetrisk strømning i KV, injiserer vi fluorescerende mikroperler i KV lumen og ta perle bevegelse ved hjelp videomicroscopy 2. Fluidstrømmen er lett visualisert og kan kvantifiseres ved å spore perle fortrengning over tid. Her, ved hjelp av scenen-spesifikk DFC målrettede genet knockdown teknikk og injeksjon av fluorescerende mikroperler inn KV å visualisere flyten presenterer vi en protokoll som gir en effektiv tilnærming for å karakterisere rollen av en bestemt gen under KV utvikling og funksjon.

Stage-spesifikke MO injeksjoner gi en nyttig tilnærming å analysere genfunksjon i bestemte avdelinger av embryoet. Figur 1 viser et flytskjema over de injeksjon strategier benyttes for å teste genfunksjon i DFC / KV celler og hvordan å introdusere fluorescerende perler å visualisere fluidstrømning i KV. Fordelingen av fluorescerende MO i vellykkede stadium-spesifikke injisert embryoer er vist skjematisk i figurene 1D-F og i levende embryoer i Figur 2. En mislykket...

Watch this Scientific Journal Video about Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle at JoVE.com

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

Cilia-genererte strømning i Kupffer sin Vesikkel (KV) styrer venstre-høyre mønster av sebrafisk embryo. Her beskriver vi en teknikk å modulere genfunksjon spesifikt i kV celler. I tillegg viser vi hvordan å levere fluorescerende perler inn KV å visualisere fluidstrømning.

Analyse av gen-funksjon og visualisering av Cilia-generert Væskestrøm i Kupffer sin Vesikkel

Internal organs such as the  heart, brain, and gut develop left-right (LR) asymmetries that are critical for  their normal functions1. Motile cilia are ...

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