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

Wij danken Fiona Foley voor uitstekende lab ondersteuning en zebravis zorg. Dit werk werd ondersteund een AHA predoctorale beurs GW (11PRE5730027) en NHLBI subsidies aan HJY (R01HL66292) en JDA (R01HL095690).

Overzicht van Stage-specifieke zebravisembryo Injecties Antisense oligonucleotiden morfolino (MO), die binden aan een doelwit-mRNA en eiwitexpressie verstoren van die transcript, worden veel gebruikt in gen knockdown (loss-of-function) studies in zebravis 13,14. Gene Tools, LLC biedt MO&#39;s die zijn voorzien van tag met ofwel carboxyfluoresceïne (zendt groene fluorescentie) of lissamine (zendt rode fluorescentie) om MO te detecteren in geïnjecteerde embryo&#39;s met behulp van fl...

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Met behulp van fase-specifieke injecties om MO te richten op de DFC / KV cellijn is een bruikbare benadering van de cel-autonomie van gen-functie te bestuderen en te voorkomen dat pleiotrope fenotypes veroorzaakt door de opwarming gen knockdown. Toch kunnen deze injecties zijn technisch uitdagend. Injectie van MO tussen de 256-cel en 1.000 cellen fasen kan resulteren in drie mogelijkheden: 1) de MO blijft geaggregeerd op de injectieplaats, 2) de MO diffundeert in de dooier en gaat DFC / KV cellen of 3) de MO diffundeert...

<table border="1"><tbody><tr><td> Naam van de Reagens / Materiaal </td><td> Vennootschap </td><td> Catalogus nummer </td></tr><tr><td> Standaard besturing tagged oligo-Lissamine </td><td> Gene Tools, LLC </td><td></td></tr><tr><td> Aangepaste Rock2b morfolino oligo </td><td> Gene Tools, LLC </td><td></td></tr><tr><td> Fluoresbrite Multifluorescent 0,5 micron microsferen </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

Interne organen zoals het hart, hersenen, darm en ontwikkelen links-rechts (LR) asymmetrie die essentieel zijn voor hun normale functie 1. Beweeglijke cilia zijn betrokken bij het ​​vaststellen van LR asymmetrie in gewervelde embryo&#39;s, met inbegrip van de muis, kikker, en zebravis 2-6. Deze &#39;LR&#39; cilia genereren asymmetrische vloeistofstroom die nodig is om een geconserveerde asymmetrische Nodal (TGF-β superfamilie) signaalcascade in de linker laterale plaat mesoderm, waarvan men denkt dat LR patronen informatie voor ontwikkelende organen 7 activeren. Dus begrijpen mechanismen LR patroon, is het essentieel om genen te identificeren die de organisatie van LR gecilieerde cellen, de motiliteit en de lengte van LR cilia en hun vermogen om robuuste asymmetrische stromen genereren reguleren. In de zebravisembryo zijn LR cilia in vesicle Kupffer&#39;s (KV) 2,4,5. KV bestaat uit een enkele laag van epitheelcellen monociliateddat een met vloeistof gevulde lumen omsluiten. Fate mapping is gebleken dat KV is afgeleid van een groep van ~ 20-30 cellen bekend als dorsale voorloper cellen (DFC) die migreren op de dorsale blastoderm marge in epiboly fasen 8,9. Tijdens de vroege stadia somiet, om DFC&#39;s cluster en differentiëren tot trilharen epitheelcellen vormen KV in de tailbud van het embryo 10,11. De mogelijkheid om te identificeren en te volgen DFC-in combinatie met optische transparantie en de snelle ontwikkeling van de zebravis embryo-maken zebravis KV een uitstekend modelsysteem om LR trilharen cellen te bestuderen. Interessant, voorvaderen van de DFC / KV cellijn te behouden cytoplasmatische bruggen tussen de dooier cel tot 4 uur na de bevruchting (HPF), terwijl cytoplasmatische bruggen tussen de dooier cel en andere embryonale cellen dicht na 2 hpf 8. Maken van deze cytoplasmatische bruggen hebben we een stadium-specifieke injectie strategie morfolino oligonucle leverenotides (MO) uitsluitend DFC en knockdown de functie van een gerichte gen in deze cellen 12. Deze techniek zorgt voor chimerisch embryo&#39;s in welk gen functie wordt naar beneden geklopt in de DFC / KV afkomst ontwikkelen in het kader van een wild-type embryo. Om asymmetrische stroming analyseren KV, we injecteren fluorescerende microbolletjes in de KV lumen en opnemen kraal beweging met behulp van videomicroscopie 2. Fluïdumstroom gemakkelijk gevisualiseerd en kan worden gekwantificeerd door het volgen bead verplaatsing in de tijd. Hier, met het stadium-specifieke DFC-targetgen knockdown techniek en injectie van fluorescerende microbolletjes in KV stromen visualiseren we een protocol dat een effectieve benadering van de rol van een bepaald gen te karakteriseren tijdens KV ontwikkeling en functie biedt.

Stadiumspecifieke MO injecties geven een nuttige benadering voor genfunctie analyse in specifieke compartimenten van de embryo. Figuur 1 toont een stroomschema van de injectie strategieën om genfunctie testen DFC / KV cellen en hoe fluorescerende kralen introduceren fluïdumstroming visualiseren in KV. De verdeling van fluorescentie in MO succesvolle stadium-specifieke geïnjecteerde embryo&#39;s is schematisch weergegeven in de figuren 1D-F en levende embryo&#39;s in Figuur 2....

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-gegenereerde vloeistofstroom in Blaasjes Kupffer&#39;s (KV) regelt links-rechts patroonvorming van de zebravis embryo. We beschrijven een techniek voor het moduleren genfunctie specifiek in KV cellen. Verder tonen we hoe fluorescerende kralen leveren in KV om vloeistofstroming visualiseren.

Analyse van gen-functie en visualisatie van Cilia-gegenereerde Fluid Flow in Blaasjes Kupffer&#39;s

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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