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

Ringraziamo Fiona Foley per il sostegno e la cura eccellente laboratorio zebrafish. Questo lavoro è stato finanziato una borsa di studio predoctoral AHA a GW (11PRE5730027) e le sovvenzioni a HJY NHLBI (R01HL66292) e JDA (R01HL095690).

Panoramica delle iniezioni zebrafish Stage-specifici embrioni Oligonucleotidi antisenso morfolino (MO), che si legano ad un mRNA mirata e disturbare espressione proteica da tale trascrizione, sono ampiamente utilizzati in knockdown gene (perdita di funzione) Studi in zebrafish 13,14. Strumenti di Gene, LLC offre MO che sono contrassegnati con uno carbossifluoresceina (emette fluorescenza verde) o lissamina (emette fluorescenza rossa) per rilevare MO negli embrioni iniettati usando la...

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Utilizzo di fase-specifici iniezioni di indirizzare MO al DFC / KV linea cellulare è un approccio utile per lo studio delle cellule-autonomia della funzione del gene e di evitare fenotipi pleiotropici causati da knockdown gene globale. Tuttavia, queste iniezioni possono essere tecnicamente impegnativo. Iniezione di MO tra le fasi 256-cellula e cellula-1000 può portare a tre possibili esiti: 1) la MO rimane aggregati nel sito di iniezione, 2) si diffonde MO tutto il tuorlo ed entra DFC / KV cellule o 3) il MO si diffon...

<table border="1"><tbody><tr><td> Nome del reagente / materiale </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Standard di controllo oligo-lissamina tag </td><td> Gene Tools, LLC </td><td></td></tr><tr><td> Personalizzato Rock2b morfolino oligo </td><td> Gene Tools, LLC </td><td></td></tr><tr><td> Fluoresbrite Multifluorescent 0,5 micron Microsfere </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

Gli organi interni come il cuore, il cervello, e nell&#39;intestino sviluppare sinistra-destra (LR) asimmetrie che sono fondamentali per le loro normali funzioni 1. Ciglia mobili sono coinvolti nella creazione LR asimmetria negli embrioni dei vertebrati, tra cui mouse, rana, e zebrafish 2-6. Questi &#39;cilia LR &quot;generano flusso asimmetrico fluido che è necessario per attivare una conservata asimmetrica Nodal (TGF-β superfamiglia) di segnalazione cascata nel mesoderma piastra laterale sinistra, che è pensato per fornire informazioni patterning LR per lo sviluppo di organi 7. Così, per comprendere i meccanismi sottostanti patterning LR, è essenziale per identificare i geni che regolano l&#39;organizzazione delle cellule ciliate LR, la motilità e la lunghezza delle ciglia LR e la loro capacità di generare robusto flusso asimmetrico. In zebrafish, ciglia LR si trovano in vescicole di Kupffer (KV) 2,4,5. KV è costituito da un singolo strato di cellule epiteliali monociliatedche racchiudono un lume piene di liquido. Mappatura destino KV ha dimostrato che è derivato da un gruppo di ~ 20-30 cellule conosciute come cellule precursore dorsali (DFCS) che migrano al margine blastoderm dorsale durante fasi epiboly 8,9. Durante le fasi iniziali somite, cluster DFCS e differenziarsi in cellule epiteliali ciliate per formare KV nel tailbud dell&#39;embrione 10,11. La capacità di identificare e tenere traccia di DFCS-in combinazione con trasparenza ottica e il rapido sviluppo del pesce zebra dell&#39;embrione-make zebrafish KV un sistema ottimo modello per studiare le cellule ciliate LR. È interessante notare che, progenitori della DFC / KV linea cellulare mantenere ponti citoplasmatici tra la cellula uovo fino a 4 ore post-fecondazione (HPF), mentre ponti citoplasmatici tra la cellula uovo e altre cellule embrionali vicino dopo 2 HPF 8. Approfittando di questi ponti citoplasmatici, abbiamo sviluppato una fase specifica strategia di iniezione per fornire morfolino oligonucleotides (MO) esclusivamente a DFCS smontabili e la funzione di un gene mirato in queste cellule 12. Questa tecnica crea embrioni chimerici in cui viene buttato funzione genica giù nella DFC / KV lignaggio sviluppare nel contesto di un embrione wild-type. Per analizzare il flusso asimmetrico fluido in KV, iniettiamo microsfere fluorescenti nel lume KV e movimento del disco perlina con videomicroscopia 2. Flusso del fluido è facilmente visualizzato e può essere quantificata inseguimento spostamento tallone nel tempo. Qui, con lo stadio-specifico DFC-targeting tecnica knockdown gene e iniezione di microsfere fluorescenti in KV per visualizzare il flusso, presentiamo un protocollo che fornisce un approccio efficace per caratterizzare il ruolo di un particolare gene durante lo sviluppo e la funzione KV.

Fase-specifici iniezioni MO fornire un approccio utile per analizzare la funzione genica in specifici compartimenti dell&#39;embrione. Figura 1 presenta un diagramma di flusso delle strategie di iniezione utilizzato per testare la funzione del gene in DFC / KV cellule e come introdurre perline fluorescenti per visualizzare il flusso del fluido in KV. La distribuzione di MO fluorescente successo in embrioni iniettati fase-specifici è mostrato schematicamente nelle figure 1D-F e in embri...

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 generato il flusso del fluido in vescicole di Kupffer (KV) controlla sinistra-destra patterning dell&#39;embrione zebrafish. Qui, si descrive una tecnica per modulare la funzione del gene specificamente in cellule KV. Inoltre, mostriamo come fornire perline fluorescenti in KV per visualizzare il flusso del fluido.

Analisi della funzione del gene e visualizzazione di Cilia-Generated flusso di fluido in vescicole di Kupffer

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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