Ringraziamo il Dr. Doris K. Wu per plasmidi di espressione e di sonde in situ, la Johns Hopkins University Center for sensoriale struttura di imaging Biologia e il Centro di udito e l&#39;equilibrio. Questo lavoro è stato sostenuto da NIDCD di Grant T32 DC000023 a LE

Le uova non schiuse e gli embrioni sono curati e trattati eticamente e umanamente. Tutti i protocolli per l&#39;uso degli embrioni unhatched sono stati approvati dalla cura degli animali e del Comitato Usa presso la Johns Hopkins School of Medicine, Baltimora, Maryland. 1. Le uova e la preparazione di costrutti di espressione <ol><li> Uova: <ol><li> Incubare 3-4 dozzina di uova di gallina fecondate (Gallus gallus) disteso su un fianco a 37 ° C. <ol><li> Dopo 24 ore di incubazione, tirare 3 cc di albume con una siringa dal back-end round del uovo e sigillare il foro con nastro adesivo trasparente. Questo crea una camera d&#39;aria tra l&#39;embrione e il guscio, consentendo facilità di accesso all&#39;embrione senza l&#39;embrione attaccare allo scafo durante il taglio la finestra di accesso sulla parte superiore dell&#39;uovo 19. </li><li> A 117 ore di tempo di incubazione, in scena gli embrioni in base alla Hamburger e Hamilton 20 fasi di sviluppoa E4 (HH24-25) (Vedi Figura 1D). </li></ol></li></ol></li><li> Espressione crea: <ol><li> Preparare il DNA plasmide utilizzando reagenti e il protocollo fornito dal produttore di un kit di preparazione disponibile in commercio. Nella fase finale di preparazione, eluire il DNA in 500 microlitri TE (Tris-EDTA) tampone fornito nel kit in una provetta da 1,5 ml. </li><li> Etanolo precipitare il DNA di concentrare aggiungendo 50 ml di 3 M acetato di sodio e 1 ml di etanolo al 100% al tubo. Posizionare il tubo a -20 ° CO / N. </li><li> Centrifugare la provetta per 30 min a 14.000 rpm a 4 ° C. Rimuovere il surnatante e aria asciugare il pellet per 10 minuti a RT. Sciogliere il pellet in 15 microlitri di buffer TE, che dovrebbe ottenere una concentrazione di ~ 4 mg / mL. NOTA: Il controllo costrutti di espressione usati qui sono pMES-IRES-GFP 10, che è guidato da un promotore di pollo β-actina, o PCI-H2B-IRES-RFP <sup&gt; 10, che è guidato dal promotore CMV. </li></ol></li></ol> 2. Pollo In Ovo Micro-elettroporazione e In Ovo Cell Proliferation Assay <ol><li> In Ovo micro-iniezione di Construct espressione e Micro-elettroporazione: <ol><li> Tirare i tubi di vetro capillari in aghi sottili con un estrattore di micro-pipetta con i seguenti parametri ottimizzati per un x 2,5 mm Box Platinum riscaldamento filamento 2.5: Pressione = 500, Calore = 600 ° C, Pull = 64, Velocity = 105, e ora = 150 msec (vedi tabella 1). </li><li> Impostare la sinistra e fasi di micro-manipolatore di mano destra che fiancheggiano il microscopio (vedi figura 1A). Il micro-manipolatore Destro tiene il capillare ago di vetro e la sinistra-handed micro-manipolatore mantiene gli elettrodi (Figura 1A). </li><li> Preparare l&#39;ago capillare di vetro finemente tirato per la micro-iniezione tagliandola punta dell&#39;ago con una pinza mentre lavora sotto il microscopio. </li><li> Riempire l&#39;ago manualmente a mano con il micro-manipolatore con 2,5 ml di DNA plasmidico colorati con 0,1% veloce verde mentre lavora sotto il microscopio. </li><li> Mettere l&#39;uovo sdraiata su un fianco all&#39;interno di un dispositivo di stampo / uovo partecipazione (vedi Figura 1A). Tagliare una finestrella sulla parte superiore dell&#39;uovo con le forbici 19 (Figura 1A). </li><li> Mentre si lavora sotto il microscopio, aprire con cautela le due membrane sovrapposte l&#39;embrione con pinze. </li><li> Lavorando al microscopio, fornire circa ~ 0,5 ml di DNA plasmidico per il giusto lume vescicola otic da micro-iniezione manualmente utilizzando la mano destra utilizzando micro-manipolatore e micro-elettroporazione. <ol><li> Per fare ciò, micro-iniettare la giusta lume vescicole otic con il DNA con la mano destra micro-manipolatore (Figura 1B-D), mentre allo stesso tempo con la mano sinistra Holdinga pinza per stabilizzare la testa dell&#39;embrione, perché la testa dell&#39;embrione immerge nella fase E4. Non micro-iniezione passato lume vescicole otic, perché questo potrebbe danneggiare la vescicola otica. </li><li> Subito dopo micro-iniezione mentre lavora sotto il microscopio, utilizzare la mano sinistra micromanipolatore per posizionare il positivo (anodo) 2 millimetri elettrodo di platino antero-ventrale vescicola otica destra e 2 millimetri negativo elettrodo (catodo) in parallelo 1 mm l&#39;uno dall&#39;altro (vedi Figura 1 C). Invia 4 impulsi a 12 V con durata di 100 msec e 200 msec spaziatura. La vescicola otica sinistra serve come un controllo non trattato interno. NOTA: Pulire gli elettrodi tra electroporations con un batuffolo di cotone imbevuto con punta in 1x PBS. </li></ol></li></ol></li><li> In Ovo Cell Proliferation Assay: <ol><li> Subito dopo l&#39;elettroporazione aggiungere 50 ml di 0,25 mg / ml EdU in 1x PBS facendo cadere manualmente la soluzione di 50 microlitri EdU su tuttaembrione in ovo con una pipetta. Sigillare le uova con nastro e tornare alla incubatore per 18-96 ore. </li><li> Rimuovere con attenzione il nastro e verificare gli embrioni per la GFP fluorescenza o il segnale di richiesta di offerta all&#39;interno della vescicola otica dopo 18 - 24 ore utilizzando un microscopio a fluorescenza standard (vedi Figura 1 EE &#39;). Se segnale fluorescente è presente, a questo punto, raccogliere gli embrioni per analisi o richiudere con nastro adesivo e restituirlo al incubatore per la raccolta ed analisi in fasi successive (vedi Figura 1 FP &#39;&#39;). NOTA: L&#39;espressione di segnale GFP con il costrutto pMES-IRES-GFP è evidente 6-7 ore dopo l&#39;elettroporazione 10. </li></ol></li></ol> 3. Embrione la raccolta e la lavorazione dei tessuti per l&#39;RNA ibridazione in situ e immunoistochimica <ol><li> Raccogliere gli embrioni con pinze. Lavare l&#39;embrione in 1x PBS freddo. Rimuovere le teste degli embrioni, tagliando la testa per il collousando pinze, e posizionare le teste in paraformaldeide al 4% O / N a 4 ° C. Perforare il cervello utilizzando una pinza per consentire la penetrazione del paraformaldeide 4% all&#39;interno della testa. </li><li> Disidratare le teste di 30% di saccarosio (in 1x PBS) O / N a 4 ° C. </li><li> Montare le teste a medio crioprotettiva da congelamento rapido in un impasto di ghiaccio secco e metilbutano (2-metilbutano). In alternativa, rapida congelare le testine in media crioprotettiva con azoto liquido. Conservare le teste montate a -80 ° C. </li><li> Cryosection le teste in 12 sezioni micron di tessuto di spessore e raccogliere tutte le sezioni di tessuto dell&#39;orecchio contenenti interno su vetrini da microscopio superfrosted. </li><li> Eseguire RNA standard di ibridazione in situ e immunoistochimica come precedentemente descritto 4,21, e analizzare per la proliferazione cellulare EdU seguendo il protocollo fornito dal produttore del kit. NOTA: le cellule dei capelli possono essere identificati utilizzando il coniglio policlonali α-MyosinVIIa (MYO7A) untibody (1: 1.000, Proteus Biosciences) e anticorpo secondario fluorescenza marcata (1: 250, di capra anti-coniglio AlexaFluor 488; Invitrogen). </li></ol> 4. Acquisizione Immagine e lavorazione <ol><li> Prendere le immagini con apparecchiature di imaging digitale. Risultati Immagine immunoistochimica con un microscopio a fluorescenza standard ed i risultati dei controlli in loco utilizzando un microscopio a livello di campo standard (che vanno da 5x a 40x in un ingrandimento). </li><li> Elaborare le immagini utilizzando il software di elaborazione delle immagini. </li></ol>

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+neurogenesis+in+the+inner+ear+requires+inhibition+of+Sox2+transcription+by+neurogenin1+and+neurod1.">Progression of neurogenesis in the inner ear requires inhibition of Sox2 transcription by neurogenin1 and neurod1.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 33, 3879-3890 (2013).</li><li>Freeman, S., Chrysostomou, E., Kawakami, K., Takahashi, Y., Daudet, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tol2-mediated+gene+transfer+and+in+ovo+electroporation+of+the+otic+placode:+a+powerful+and+versatile+approach+for+investigating+embryonic+development+and+regeneration+of+the+chicken+inner+ear.">Tol2-mediated gene transfer and in ovo electroporation of the otic placode: a powerful and versatile approach for investigating embryonic development and regeneration of the chicken inner ear.</a> Methods in molecular biology. 916, 127-139 (2012).</li><li>Kamaid, A., Neves, J., Giraldez, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Id+gene+regulation+and+function+in+the+prosensory+domains+of+the+chicken+inner+ear:+a+link+between+Bmp+signaling+and+Atoh1.">Id gene regulation and function in the prosensory domains of the chicken inner ear: a link between Bmp signaling and Atoh1.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 30, 11426-11434 (2010).</li><li>Neves, J., Kamaid, A., Alsina, B., Giraldez, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+Sox2+and+Sox3+in+neuronal+and+sensory+progenitors+of+the+developing+inner+ear+of+the+chick.">Differential expression of Sox2 and Sox3 in neuronal and sensory progenitors of the developing inner ear of the chick.</a> The Journal of comparative neurology. 503, 487-500 (2007).</li><li>Neves, J., Uchikawa, M., Bigas, A., Giraldez, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prosensory+function+of+Sox2+in+the+chicken+inner+ear+relies+on+the+direct+regulation+of+Atoh1.">The prosensory function of Sox2 in the chicken inner ear relies on the direct regulation of Atoh1.</a> PLoS One. 7, 30871 (2012).</li><li>Das, R. 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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+non-small+cell+lung+tumor+development+by+the+let-7+microRNA+family.">Suppression of non-small cell lung tumor development by the let-7 microRNA family.</a> Proc Natl Acad Sci U S A. 105, 3903-3908 (2008).</li><li>Jacques, B. E., Dabdoub, A., Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgf+signaling+regulates+development+and+transdifferentiation+of+hair+cells+and+supporting+cells+in+the+basilar+papilla.">Fgf signaling regulates development and transdifferentiation of hair cells and supporting cells in the basilar papilla.</a> Hearing research. 289, 27-39 (2012).</li><li>Korn, M. J., Cramer, K. 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The authors have nothing to disclose.

Il metodo qui descritto di in ovo micro-elettroporazione è ottimizzato per il trasferimento genico in via di sviluppo dell&#39;organo uditivo. E &#39;compatibile con il plasmide vettori di espressione basati sul DNA tipicamente utilizzati per manipolare la funzione del gene / espressione. I tempi di elettroporazione in E4 è ottimale per le indagini che si concentrano sullo sviluppo HC nell&#39;organo uditivo. Le fasi più critiche sono micro-iniezione del DNA nel lume vescicola otic senza andare troppo in pro...

Nonostante le differenze nella morfologia e patterning cellulare tra mammiferi e aviaria organo sensoriale uditivo, i fattori molecolari e dei percorsi responsabili dello sviluppo HC sensoriali sono pensati per essere evolutivamente conservato 1-3. La papilla basilare, che ospita le HC uditive e le loro SC circostanti, si sviluppa come un outpocketing del otocyst dell&#39;orecchio interno. Nella fase iniziale di un pool di progenitori HC e SC è specificato all&#39;interno del / otic dominio competente tazza neurale-sensoriale otic placode (NSD). Fate dati di mappatura dimostrano che lignaggi neuronali e sensoriali sono collegati e nascono dalla NSD situato nella regione antero-ventrale della coppa otic o otocyst nei topi e pollo 4,5. In primo luogo, neuroblasti delaminano dal NSD per dare origine ai neuroni del ganglio uditivo-vestibolare, che alla fine diviso in gangli uditivo e vestibolare. Questi neuroni innervano i HC sensoriali dell&#39;orecchio interno e nuclei del tronco cerebrale. Le cellule Tha rimanere nel NSD si pensa di dare origine a varie patch sensoriali, tra cui l&#39;organo sensoriale uditivo, costituito dai HC sensoriali meccano-trasduzione e le loro SC associati. In entrambi il pulcino e murino, organo uditivo progenitrice sensoriale uscita del ciclo cellulare e la differenziazione HC si verificano in opposte gradienti. In pulcino, uditivo progenitrice uscita del ciclo cellulare inizia intorno ~ E5 e progredisce dalla base all&#39;apice, e dal centro alla periferia 6. Il giorno dopo, la differenziazione HC inizia nel vertice e progredisce alla base 7. Studiando lo sviluppo dell&#39;orecchio interno nei polli offre molti vantaggi tecnici meccanismi funzionali possono essere studiate con relativa facilità in ovo al contrario di manipolare embrioni in utero in altri sistemi modello, che richiede interventi complessi. Il metodo descritto qui utilizza in ovo micro-elettroporazione per indirizzare i geni di interesse nella Basila presuntivaR zona della papilla antero-ventrale nella vescicola otica specificamente a E4. Elettroporazione in ovo è una tecnica che è ben consolidata e comunemente usato 8-14. Il principio della tecnica elettroporazione si basa sul fatto che i nucleotidi (ad esempio, DNA plasmidico, DNA sintetico, o RNA oligonucleotidi) sono caricati negativamente. Il DNA viene iniettato nel tessuto di interesse. Quando una corrente elettrica viene inserito attraverso il tessuto, la corrente si apre pori transitori nelle pareti cellulari e permette l&#39;assorbimento del DNA, come il DNA caricato negativamente fluisce verso l&#39;elettrodo positivo (anodo). Per gli esperimenti di guadagno di funzione, i geni di interesse sono comunemente subclonati in plasmidi di espressione che contengono adeguate cassette di espressione per Green Fluorescent Protein (GFP) o proteina fluorescente rossa (RFP). Per la perdita di funzione esperimenti plasmide-based dominanti costrutti negativi repressori, o RNAi, o costrutti morpholino sono commonly usato 15,16. Per inibire microRNA (miRNA) funzione miRNA costrutti spugna, che sono tipicamente plasmide base, può essere utilizzato 17. Il metodo qui descritto permette di indagare i meccanismi molecolari che controllano la proliferazione e la differenziazione nell&#39;organo uditivo, come nel metodo di micro-elettroporazione ovo possono essere facilmente combinati con in test di proliferazione cellulare ovo aggiungendo EdU a tutta dell&#39;embrione. L&#39;elettroporazione e l&#39;aggiunta di EdU viene eseguita a E4 nella vescicola otica, che è un giorno prima della comparsa di uscita del ciclo cellulare nell&#39;organo uditivo. L&#39;analisi dell&#39;organo uditivo è in genere eseguita 18-96 ore dopo l&#39;elettroporazione in E5 (insorgenza di uscita del ciclo cellulare progenitrici sensoriale), E6 (insorgenza di differenziazione HC), e E7 (durante la differenziazione HC); e poi fino a ~ E9 (fine differenziazione HC). Questo metodo elettroporazione del trasferimento genico è transitorio della durata di circa ~ 4 giorni, because i geni di interesse non si integrano nel genoma, ma il metodo è applicabile per l&#39;uso con adeguato plasmide vettori di espressione basati sul DNA, che hanno la capacità di integrarsi nel genoma, come ad esempio il trasferimento genico mediato Tol2 11. Con questo metodo GFP robusto o espressione RFP dura ~ 4 giorni nella coclea, dopo che indicano i segnali di dissolvenza, ma forniscono una finestra di tempo sufficiente per studiare lo sviluppo intricato dell&#39;organo uditivo. Questo metodo di trasferimento genico in ovo è nuovo e consente di indirizzare specificamente l&#39;organo uditivo presuntiva in E4, che è ottimale per le indagini che si concentrano sullo sviluppo HC nell&#39;organo uditivo. Si tratta di una buona aggiunta ai metodi alternativi, che l&#39;elettroporazione in molto più giovani stadi di sviluppo o 11,12 rispetto all&#39;uso di in vitro basilare papille culture espianto 18.

<table border="0" cellpadding="0" cellspacing="0" width="1129">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:621px;">Sterile 1x PBS pH 7.4</td>
			<td style="width:249px;">gibco</td>
			<td style="width:259px;">10010-023</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast Green FCF Powder</td>
			<td>Sigma</td>
			<td>F7252-5G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EdU Powder</td>
			<td>Invitrogen</td>
			<td>E10187</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Click-IT EdU Alexa Fluor 555 Imaging Kit</td>
			<td>Invitrogen</td>
			<td>C10338</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HiSpeed Plasmid Midi Kit (25)</td>
			<td>Qiagen</td>
			<td align="right">12643</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ECM 830 ElectroSquarePorator&#160;</td>
			<td>BTX Harvard Apparatus</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Banana to Micrograbber Cable Kit</td>
			<td>BTX Harvard Apparatus</td>
			<td>45-0216</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Right Handed &#38; Left Handed Micromanipulators</td>
			<td>World Precision Instruments Inc.&#160;</td>
			<td>M3301R &#38; M3301L</td>
		</tr>
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			<td height="21" style="height:21px;">Two 12 mm Magnetic Holding Device Stages with 7 inch vertical posts</td>
			<td>World Precision Instruments Inc.&#160;</td>
			<td>M10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Metal Steel Base Plate 12x24 inch</td>
			<td>World Precision Instruments Inc.&#160;</td>
			<td align="right">5479</td>
		</tr>
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			<td height="21" style="height:21px;">Scissors for Eggs 12 cm long curved, 12 mm extrafine blades; Spring Scissors</td>
			<td>World Precision Instruments Inc.&#160;</td>
			<td align="right">14120</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropippette Puller for pulling needles</td>
			<td>Sutter Instrument Co.</td>
			<td>P-97</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2.5x2.5 mm Box Platinum Heating Filament</td>
			<td>Sutter Instrument Co.</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Glass Capillary Tubes/Needles/No Fiber/Borosil 1 mm</td>
			<td>FHC</td>
			<td>27-30-0</td>
		</tr>
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			<td height="21" style="height:21px;">Hamilton Glass Syringe 100 &#956;l</td>
			<td>Hamilton</td>
			<td>80601 Model 710LT</td>
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			<td height="21" style="height:21px;">Mineral Oil (Heavy) for Hamilton Glass Syringe</td>
			<td>Fisher Scientific</td>
			<td>O122-1</td>
		</tr>
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			<td height="21" style="height:21px;">Polyethylene Tubing for connecting the Glass Syringe and Glass Cappillary Needles/ Non Toxic</td>
			<td>Becton Dickinson and Company (BD)</td>
			<td>427420 Intramedic Clay Adams Brand</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3 cc Disposable Syringes</td>
			<td>Becton Dickinson and Company (BD)</td>
			<td align="right">309657</td>
		</tr>
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			<td height="21" style="height:21px;">Disposable 21 Gauge Needles</td>
			<td>Becton Dickinson and Company (BD)</td>
			<td align="right">305122</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">One pair of 2 mm Platinum Electrodes</td>
			<td>Bulldog Bio. / Nepagene</td>
			<td>CUY611P3-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electrode Holder</td>
			<td>Bulldog Bio. / Nepagene</td>
			<td>CUY580</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">One pair of Dumont fine forceps number 5</td>
			<td>Fine Science Tools (FST)</td>
			<td></td>
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			<td height="21" style="height:21px;">Matte finish invisible tape for sealing eggs</td>
			<td>Office Depot</td>
			<td>520-928</td>
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			<td height="21" style="height:21px;">Cotton-Tipped Swabs</td>
			<td>Fisher Scientific</td>
			<td>23-400-101</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile filter tips</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

gene transfer into the chicken auditory organ by in ovo micro-electroporation

Embrioni di pollo sono sistemi modello ideale per lo studio dello sviluppo embrionale, come manipolazioni di funzione del gene possono essere svolte con relativa facilità in ovo. L&#39;orecchio interno uditiva organo sensoriale è fondamentale per la nostra capacità di sentire. Ospita un epitelio altamente specializzato sensoriale che è costituito da cellule ciliate meccano-trasduzione (HC) e dintorni gliali-come le cellule di supporto (SCS). Nonostante le differenze strutturali negli organi uditivi, meccanismi molecolari che regolano lo sviluppo dell&#39;organo uditivo sono evolutivamente conservati tra mammiferi e aves In elettroporazione Ovo è in gran parte limitata alle prime fasi a E1 -. E3. A causa della relativa ritardato sviluppo dell&#39;organo uditivo a E5, manipolazioni dell&#39;organo uditivo di elettroporazione in ovo passato E3 sono difficili a causa dello sviluppo advanced dell&#39;embrione di pollo in fasi successive. Il metodo qui presentato è un metodo di trasferimento genico transitoria per il targeting dei geni di interesse afase E4 - E4.5 in via di sviluppo pollo uditivo organo sensoriale via in ovo micro-elettroporazione. Questo metodo è applicabile per GAIN- e perdita di funzioni con plasmide convenzionale vettori di espressione basati sul DNA e può essere combinato con in saggio di proliferazione delle cellule ovo aggiungendo EdU (5-etinil-2&#39;-deossiuridina) per l&#39;intero embrione allo tempo di elettroporazione. L&#39;uso di proteina fluorescente verde o rosso (GFP o RFP) plasmidi di espressione consente allo sperimentatore di determinare rapidamente se l&#39;elettroporazione di mira con successo la porzione uditiva del sviluppo dell&#39;orecchio interno. In questo lavoro il metodo, esempi rappresentativi di esemplari GFP elettroporate sono illustrate; embrioni sono stati raccolti 18-96 ore dopo l&#39;elettroporazione e il targeting della GFP per l&#39;area pro-sensoriale dell&#39;organo uditivo è stata confermata da RNA ibridazione in situ. Il documento metodo fornisce anche un protocollo ottimizzato per l&#39;uso della timidina analogico EdU analizzare prolif cellulerazione; un esempio di un saggio di proliferazione cellulare basato EdU che combina immuno-etichettatura e cliccare EdU chimica è fornito.

In questo DNA metodo della carta plasmide composto di proteina fluorescente verde (GFP) cassette di espressione è stata mirata al pollo in via di sviluppo papilla basilare (BP) con parametri ottimizzati da 12 V e 4 impulsi con 100 durata degli impulsi msec e gli intervalli di 200 msec, ottenendo un ~ tasso di sopravvivenza del 50% degli embrioni e l&#39;efficienza del plasmide-DNA mira alla BP. Imaging fluorescente di espressione GFP nativa in embrioni di mostrato questo metodo di elett...

Watch this Scientific Journal Video about Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation at JoVE.com

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Gene Transfer nel pollo Auditory Organ<em&gt; In Ovo</em&gt; Micro-elettroporazione

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

gene-transfer-into-chicken-auditory-organ-ovo-micro

Research

JoVE Journal

Developmental Biology

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

9.4K Views.  The Johns Hopkins University, School of Medicine. The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ. 

Video: Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

Trasferimento efficiente Gene in Chick retine per cellulari primarie Cultura Studies: An<em&gt; Ex-ovo</em&gt; Elettroporazione Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

Ricerca

Biologia dello sviluppo

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Cultura della embrionali di topo cocleari espianti e Gene Transfer con elettroporazione

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

Metodi di trasferimento Proprio localizzata di cellule o DNA in precoce post-impianto di embrioni di topo

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression and activation of signal transduction pathways. This technique allows the delivery of signaling molecules at precisely defined developmental stages for specific times. After this embryos can be harvested and gene expression examined, for example by in situ hybridization, or activation of signal transduction pathways observed with immunostaining.
In this video heparin beads soaked in FGF18 or AG 1-X2 beads soaked in U0126, a MEK inhibitor, are grafted into the limb bud in ovo. This shows that FGF18 induces expression of MyoD and ERK phosphorylation and both endogenous and FGF18 induced MyoD expression is inhibited by U0126. Beads soaked in a retinoic acid antagonist can potentiate premature MyoD induction by FGF18.
This approach can be used with a wide range of different growth factors and inhibitors and is easily adapted to other tissues in the developing embryo.

By grafting beads soaked in growth factors or specific inhibitors of signaling pathways into developing embryos it is possible to directly test their effects in vivo. In this protocol beads are grafted into the limb bud to determine the effects of these molecules on gene expression and signal transduction.

L&#39;innesto di Perline in via di sviluppo di pollo Limbs embrioni per identificare percorsi di trasduzione del segnale che influenzano l&#39;espressione genica

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression ...

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and molecular level. To alter intracardiac hemodynamics, fertilized chicken eggs are incubated in a humidified chamber to obtain embryos of the desired stage (HH17). Once this developmental stage is achieved, the embryo is maintained ex ovo and hemodynamics in the embryonic heart are altered by partially constricting the outflow tract (OFT) with a surgical suture at the junction of the OFT and ventricle (OVJ). Control embryos are also cultured ex ovo but are not subjected to the surgical intervention. Banded and control embryos are then incubated in a humidified incubator for the desired period of time, after which 2D ultrasound is employed to analyze the change in blood flow velocity at the OVJ as a result of OFT banding. Once embryos are maintained ex ovo, it is important to ensure adequate hydration in the incubation chamber so as to prevent drying and eventually embryo death. Using this new banded model, it is now possible to perform analyses of changes in the expression of key players involved in valve development and to understand the role of hemodynamics on cellular responses in vivo, which could not be achieved previously.

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

For the first time we present here a reproducible banding procedure to alter hemodynamics in the developing heart ex ovo. This is achieved by partially constricting the outflow tract (OFT).

Un romanzo<em&gt; Ex Ovo</em&gt; Tecnica Banding alterare intracardiaca Emodinamica in un sistema embrionali di pollo

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and ...

In Ovo Electroporation in the Chicken Auditory Brainstem

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that the chicken embryo is an effective research model for studying basic biological functions of auditory system development. More recently, the chicken embryo has become particularly valuable in studying gene expression, regulation and function associated with hearing. In ovo electroporation can be used to target auditory brainstem regions responsible for highly specialized auditory functions. These regions include the chicken nucleus magnocellularis (NM) and nucleus laminaris (NL). NM and NL neurons arise from distinct precursors of rhombomeres 5 and 6 (R5/R6). Here, we present in ovo electroporation of plasmid-encoded genes to study gene-related properties in these regions. We show a method for spatial and temporal control of gene expression that promote either gain or loss of functional phenotypes. By targeting auditory neural progenitor regions associated with R5/R6, we show plasmid transfection in NM and NL. Temporal regulation of gene expression can be achieved by adopting a tet-on vector system. This is a drug inducible procedure that expresses the genes of interest in the presence of doxycycline (Dox). The in ovo electroporation technique &#8211; together with either biochemical, pharmacological, and or in vivo functional assays &#8211; provides an innovative approach to study auditory neuron development and associated pathophysiological phenomena.

In Ovo Electroporation in the Chicken Auditory Brainstem

Auditory brainstem neurons of avians and mammals are specialized for fast neural encoding, a fundamental process for normal hearing functions. These neurons arise from distinct precursors of embryonic hindbrain. We present techniques utilizing electroporation to express genes in the hindbrain of chicken embryos to study gene function during auditory development.

In Ovo Electroporation nel Brainstem Auditory del pollo

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

CRISPR-mediata di perdita di funzione di analisi in granuli cerebellari cellule utilizzando nell'Utero basati su elettroporazione trasferimento del Gene

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

Colture organotipiche tridimensionale degli organi sensoriali uditivi e vestibolari

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The continuously growing mouse incisor provides an excellent model to study tissue-specific stem cells. The epithelial tissue-specific stem cells of the incisor are located at the proximal end of the tooth in a niche called the cervical loop. They provide a continuous influx of cells to counterbalance the constant abrasion of the self-sharpening tip of the tooth. Presented here is a detailed protocol for the isolation and culture of the proximal end of the mouse incisor that houses stem cells and their niche. This is a modified Trowell-type organ culture protocol that enables in vitro culture of tissue pieces (explants), as well as the thick tissue slices at the liquid/air interface on a filter supported by a metal grid. The organ culture protocol described here enables tissue manipulations not feasible in vivo, and when combined with the use of a fluorescent reporter(s), it provides a platform for the identification and tracking of discrete cell populations in live tissues over time, including stem cells. Various regulatory molecules and pharmacological compounds can be tested in this system for their effect on stem cells and their niches. This ultimately provides a valuable tool to study stem cell regulation and maintenance.

The Trowell-type organ culture method has been used to unravel complex signaling networks that govern tooth development and, more recently, for studying regulation involved in stem cells of the continuously growing mouse incisor. Fluorescent-reporter animal models and live-imaging methods facilitate in-depth analyses of dental stem cells and their specific niche microenvironment.

Uso della coltura di organi di tipo Trowell per studiare la regolazione delle cellule staminali dentali

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

Isolamento delle cellule staminali mesenchimali del tessuto adiposo di ratto per la differenziazione in cellule produttrici di insulina

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...

Gene Transfer nel pollo Auditory Organ

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Trasferimento efficiente Gene in Chick retine per cellulari primarie Cultura Studies: An