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 fremragende lab support og zebrafisk pleje. Dette arbejde blev støttet af en AHA predoctoral stipendium til GW (11PRE5730027) og NHLBI tilskud til HJY (R01HL66292) og JDA (R01HL095690).

Oversigt over Stage-specifikke zebrafisk Embryo Indsprøjtninger Antisense morpholino oligonucleotider (MO), der binder til en målrettet mRNA og forstyrre proteinekspression fra dette transkript, er meget udbredt i gen knockdown (tab-af-funktion) undersøgelser i zebrafisk 13,14. Gene Tools, LLC tilbyder MOs, der er mærket med enten carboxyfluorescein (udsender grøn fluorescens) eller lissamin (udsender rød fluorescens) til påvisning MO i injicerede embryoner ved hjælp af fluor...

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Brug af fase-specifikke injektioner at målrette MO til DFC / KV celleafstamning er en nyttig tilgang til at studere celle-autonomi genfunktion og undgå pleiotrope fænotyper som følge af den globale gen knockdown. Dog kan disse injektioner være teknisk udfordrende. Injektion af MO mellem de 256-celle-og 1.000-celle faser kan resultere i tre mulige udfald: 1) den MO forbliver aggregeres på injektionsstedet, 2) de MO diffunderer hele blommen og kommer ind DFC / KV celler eller 3) den MO diffunderer gennem hele blomme...

<table border="1"><tbody><tr><td> Navn på reagens / Material </td><td> Firma </td><td> Katalognummer </td></tr><tr><td> Standard Kontrol mærkede oligo-Lissamin </td><td> Gene Tools, LLC </td><td></td></tr><tr><td> Brugerdefineret Rock2b morpholino oligo </td><td> Gene Tools, LLC </td><td></td></tr><tr><td> Fluoresbrite Multifluorescent 0,5 um mikrosfærer </td><td> Polysciences, Inc. </td><td> 24.054 </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 tarm udvikle venstre-højre (LR) asymmetrier, der er kritiske for deres normale funktioner 1. Motile cilier er involveret i at etablere LR asymmetri i hvirveldyr embryoner, herunder mus, frø, og zebrafisk 2-6. Disse &quot;LR cilia &#39;generere asymmetriske fluidstrøm, der er nødvendig for at udløse en konserveret asymmetrisk Nodal (TGF-β superfamilien) signaleringskaskade i den venstre laterale plade mesoderm, som menes at tilvejebringe LR mønster information for at udvikle organer 7. Således at forstå mekanismerne bag LR mønsterdannelse er det vigtigt at identificere gener, der regulerer tilrettelæggelsen af ​​LR cilierede celler, motilitet og længde LR cilier og deres evne til at skabe en robust asymmetrisk strømning. I zebrafisk embryo, er LR cilia beliggende i Kupffer s vesikel (KV) 2,4,5. KV består af et enkelt lag af monociliated epithelcellerat vedlægge en væskefyldt hulrum. Fate kortlægning har vist, at KV er afledt af en gruppe ~ 20-30 celler kendt som dorsale forløberen celler (DFCs), der vandrer på den dorsale blastoderm margin under epiboly faser 8,9. Under tidlige somit faser, til DFCs klynge og differentiere til cilierede epitelceller danne KV i tailbud af embryonet 10,11. Evnen til at identificere og spore DFCs-i kombination med optisk transparens og hurtig udvikling af zebrafisk embryo-gør zebrafisk KV en fremragende model til undersøgelse LR cilierede celler. Interessant, progenitorer af DFC / KV celleafstamning bevarer cytoplasmiske broer mellem blommen celle op til 4 timer efter befrugtning (HPF), hvorimod cytoplasmiske broer mellem blommesækken celle og andre embryonale celler tæt efter 2 HPF 8. Drage fordel af disse cytoplasmiske broer, vi udviklet en trinspecifik injektion strategi om at levere morpholino oligonucleotides (MO) udelukkende til DFCs og knockdown funktionen af en målrettet gen i disse celler 12. Denne teknik skaber kimære embryoner i hvilket gen funktion er slået ned i DFC / KV afstamning udvikler sig i forbindelse med en vildtype-embryo. At analysere asymmetrisk fluidstrøm i KV, vi indsprøjte fluorescerende mikroperler i KV lumen og optage perle bevægelsen ved hjælp videomicroscopy 2. Fluidstrømning er let visualiseres og kan kvantificeres ved at spore vulst forskydning over tid. Her bruger trinspecifik DFC målrettet gen knockdown teknik og injektion af fluorescerende mikroperler i KV at visualisere flow, præsenterer vi en protokol, der giver en effektiv tilgang til at karakterisere rollen af ​​et bestemt gen i løbet af KV udvikling og funktion.

Fase-specifikke MO injektioner giver en nyttig fremgangsmåde til at analysere genfunktion i bestemte rum i embryoet. Figur 1 viser et rutediagram over injektionsanlæggene strategier anvendes til at teste genfunktion i DFC / KV celler og hvordan at indføre fluorescerende perler at visualisere fluidstrømning i KV. Fordelingen af fluorescerende MO i vellykkede fase-specifikke injicerede embryoner er vist skematisk i figur 1D-F og i levende embryoner i fig. 2. Et mislyk...

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-genereret fluidstrøm i Kupffer s vesikel (KV) styrer venstre-højre mønstret af den zebrafisk embryo. Her beskriver vi en teknik til at modulere genfunktion specifikt i KV celler. Derudover viser vi, hvordan man leverer fluorescerende perler i KV at visualisere fluidstrømning.

Analyse af genfunktioner og Visualisering af Cilia er genereret Fluid Flow i Kupffer s vesikel

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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