Wir danken Dr. Doris K. Wu für Expressionsplasmide und in - situ - Sonden, die Johns Hopkins University Center für Sinnesbiologie Bildgebung Anlage und das Zentrum für Hören und Gleichgewicht. Diese Arbeit wurde von NIDCD Grants T32 DC000023 zu LE unterstützt

Die Eier und unhatched Embryonen werden gepflegt und ethisch und menschlich behandelt. Alle Protokolle für unhatched Embryo Verwendung durch das Animal Care und Use Committee an der Johns Hopkins School of Medicine, Baltimore, Maryland genehmigt. 1. Eier und Herstellung von Expressionskonstrukten <ol><li> Eier: <ol><li> Inkubieren 3-4 Dutzend befruchtete Hühnereier (Gallus gallus) bei 37 ° C flach auf der Seite liegen. <ol><li> Nach 24 Stunden Inkubation ziehen 3 ccm Eiweiss mit einer Spritze aus dem runden hinteren Ende des Eis und das Loch mit durchsichtigem Klebeband abzudichten. Dies schafft eine Luftkammer zwischen dem Embryo und der Eierschale, so dass für einen leichten Zugang zu dem Embryo , ohne den Embryo an der Schale haftet , wenn für den Zugriff auf die Oberseite des Eies 19 das Fenster zu schneiden. </li><li> Bei 117 h Inkubationszeit, inszenieren die Embryonen nach Hamburger und Hamilton 20 Entwicklungsstadienbei E4 (HH24-25) (siehe Figur 1D). </li></ol></li></ol></li><li> Expressionskonstrukten: <ol><li> Bereiten Sie die Plasmid-DNA unter Verwendung von Reagenzien und das Protokoll vom Hersteller eines handelsüblichen Vorbereitung Kit zur Verfügung gestellt. Im letzten Schritt der Herstellung, eluieren die DNA in 500 ul TE (Tris-EDTA) -Puffer in dem Kit in ein 1,5-ml-Röhrchen bereitgestellt. </li><li> Ethanol ausfällen die DNA sie zu konzentrieren durch Zugabe von 50 ul 3 M Natriumacetat und 1 ml 100% Ethanol zu dem Rohr. Das Röhrchen wird bei -20 ° CO / N. </li><li> Zentrifugieren Sie die Röhrchen für 30 min bei 14.000 rpm bei 4 ° C. Entfernen Sie den Überstand und an der Luft trocknen das Pellet für 10 min bei RT. Auflösen des Pellets in 15 ul TE-Puffer, die eine Konzentration von ~ 4 &amp; mgr; g / &amp; mgr; l ergeben sollte. HINWEIS: Die Expressionskontroll verwendeten Konstrukte sind hier Pmes-IRES-GFP 10, die von einem Huhn β-Aktin - Promotor oder pCI-H2B-IRES-RFP angetrieben wird <sbis&gt; 10, die durch den CMV - Promotor angetrieben wird. </li></ol></li></ol> 2. Huhn In Ovo Micro-Elektroporation und in - ovo - Zellproliferationstest <ol><li> In Ovo Mikroinjektion von Expression Baukonstruktion und Micro-Elektroporation: <ol><li> Ziehen die Glaskapillaren in feinen Nadeln mit einer Mikropipettenzieher mit den folgenden optimierten Parameter für eine 2,5 x 2,5 mm Box Platinum Heizdraht: Druck = 500, Wärme = 600 ° C, ziehen = 64, Geschwindigkeit = 105, und Zeit = 150 ms (siehe Tabelle 1). </li><li> Die links- und rechtsgeben Mikromanipulator Stufen flankieren das Mikroskop (siehe Abbildung 1A) ein. Der Rechtshänder Mikromanipulator hält die Glaskapillare Nadel und der Linkshänder Mikromanipulator hält die Elektroden (1A). </li><li> Bereiten Sie die fein gezogen Glaskapillare Nadel für Mikroinjektion durch Schneidendie Spitze der Nadel mit einer Zange während der Arbeit unter dem Mikroskop. </li><li> Füllen Sie die Nadel manuell von Hand mit dem Mikromanipulator mit 2,5 ul von Plasmid-DNA mit 0,1% Fast Green getönt, während sie unter dem Mikroskop zu arbeiten. </li><li> Legen Sie das Ei auf der Seite liegend in einer Form / Ei Haltevorrichtung (siehe 1A). Schneiden Sie ein rundes Fenster auf dem Ei mit einer Schere 19 (1A). </li><li> Während unter dem Mikroskop arbeiten, öffnen vorsichtig die beiden Membranen den Embryo überlagert mit einer Pinzette. </li><li> Während unter dem Mikroskop arbeiten, liefern mit dem Mikromanipulator und durch Mikro-Elektroporation ca. ~ 0,5 ul von Plasmid-DNA an die richtigen Ohrbläschen Lumen durch Mikroinjektion in die rechte Hand. <ol><li> Dazu Mikro injizieren die richtigen Ohrbläschen Lumen mit der DNA mit der rechten Mikromanipulator (1B-D) , während zur gleichen Zeit die linke Hand Holdin mitga forcep den Kopf des Embryos zu stabilisieren, weil der Kopf des Embryos in der Stufe E4 eintaucht. Nicht Mikro injizieren vorbei an den Ohrbläschen Lumen, da dies das Ohrbläschen schädigen. </li><li> Unmittelbar nach der Mikroinjektion, während sie unter dem Mikroskop arbeiten, verwenden Sie die linke Mikromanipulator die positive (Anode) 2 mm Platinelektrode anterior-ventralen nach rechts Ohrbläschen und der negativen 2 mm Elektrode (Kathode) parallel zu platzieren 1 mm auseinander (Abbildung 1 C sehen). Geben Sie 4 Impulse bei 12 V mit 100 ms Dauer und 200 ms Abstand. Das linke Ohrbläschen dient als interne unbehandelten Kontrolle. HINWEIS: Reinigen Sie die Elektroden zwischen electroporations mit einem Wattetupfer getaucht in 1x PBS. </li></ol></li></ol></li><li> In Ovo - Zellproliferationstest: <ol><li> Unmittelbar nach der Elektroporation werden 50 ul 0,25 mg / ml EdU in 1x PBS, indem manuell die 50 ul EdU Lösung auf die gesamte droppingEmbryo in ovo mit einer Pipette. Verschließen Sie die Eier mit einem Band und zurück in den Inkubator für 18-96 Std. </li><li> Sie das Band vorsichtig entfernen und nach 18 innerhalb der Ohrbläschen , die Embryonen für Fluoreszenz GFP oder RFP Signal überprüfen - 24 Stunden ein Standard - Fluoreszenzmikroskop (siehe Abbildung 1 EE &#39;) verwendet wird . Wenn Fluoreszenzsignal vorhanden ist, ernten an dieser Stelle die Embryonen für Analysen oder mit Klebeband wieder verschließen und senden Sie es für die Ernte in den Inkubator und Analysen in späteren Phasen (Abbildung 1 FP &#39;&#39; zu sehen). HINWEIS: Die Expression von GFP - Signal mit dem PME-IRES-GFP - Konstrukt ist offensichtlich , 6 - 7 Stunden nach der Elektroporation 10. </li></ol></li></ol> 3. Embryo Ernten und Gewebeverarbeitung für die RNA in situ Hybridisierung und Immunhistochemie <ol><li> Ernten Sie die Embryonen mit einer Pinzette. Spülen Sie den Embryo in kaltem 1x PBS. Entfernen Sie die Köpfe der Embryonen, durch Schneiden der Kopf durch den Halsmit einer Pinzette und legen die Köpfe in 4% Paraformaldehyd O / N bei 4 ° C. Stechen Sie das Gehirn eine forcep mit Durchdringung der 4% Paraformaldehyd im Inneren des Kopfes zu ermöglichen. </li><li> Entwässern die Köpfe in 30% Saccharose (in 1x PBS) O / N bei 4 ° C. </li><li> Montieren Sie die Köpfe in Kryoprotektivum durch schnelles Einfrieren in einem Brei aus Trockeneis und Methylbutan (2-Methyl). Alternativ frieren schneller die Köpfe in kryoprotektiver Medium mit flüssigem Stickstoff. Lagern Sie die montierten Köpfe bei -80 ° C. </li><li> Kryoschnittes die Köpfe in 12 &amp; mgr; m dicke Gewebeschnitte und sammeln Sie alle Innenohr haltigen Gewebeschnitte auf superfrosted Mikroskop Objektträger aus Glas. </li><li> Führen Sie Standard - RNA in situ Hybridisierung und Immunhistochemie , wie zuvor beschrieben 4,21 und analysieren für EdU Zellproliferation nach dem Protokoll vom Hersteller des Kits zur Verfügung gestellt. HINWEIS: Haarzellen unter Verwendung von polyklonalen Kaninchen-α-MyosinVIIa (MYO7A) ein identifiziert werden könnentibody (1: 1000, Proteus Biosciences) und fluoreszenzmarkierter sekundärer Antikörper (1: 250, Ziege anti-Kaninchen AlexaFluor 488; Invitrogen). </li></ol> 4. Bildaufnahme- und Bildverarbeitungs <ol><li> Nehmen Sie Bilder mit digitalen Bildgeräte. Bild Immunhistochemie Ergebnisse mit einem Standard - Fluoreszenzmikroskop und die Ergebnisse der in situ ein Standardweitfeldmikroskop ( im Bereich von 5x bis 40x in Vergrßerung) gemessen. </li><li> Verarbeiten Sie die Bilder mit Hilfe von Bildverarbeitungssoftware. </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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Windowing+chicken+eggs+for+developmental+studies.">Windowing chicken eggs for developmental studies.</a> Journal of visualized experiments: JoVE. , e306 (2007).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Journal of morphology. 88, 49-92 (1951).</li><li>Oh, S. H., Johnson, R., Wu, D. 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The authors have nothing to disclose.

Das hier beschriebene Verfahren der in - ovo - Mikro Elektroporation wird für den Gentransfer in die Entwicklungs Hörorgan optimiert. Es ist kompatibel mit Plasmid-DNA basierenden Expressionsvektoren typischerweise verwendet, um die Genfunktion / Ausdruck zu manipulieren. Der Zeitpunkt der Elektroporation bei E4 ist für Untersuchungen optimal, die im Hörorgan auf HC Entwicklung konzentrieren. Die wichtigsten Schritte sind die DNA in das Ohrbläschen Lumen Mikrospritz ohne mit der Nadel geht zu tief...

Trotz der Unterschiede in der Morphologie und Zell Strukturierung zwischen Säugetieren und Vogel auditorischen Sinnesorgan, die molekularen Faktoren und Wege verantwortlich für die sensorische HC Entwicklung sind gedacht , um evolutionär 1-3 konserviert werden. Die basilar Papille, die den Gehör HCs und der sie umgebenden SCs beherbergt, entwickelt als Aussackung des Innenohrs otocyst. Früh auf einem Pool von HC und SC Vorläufern ist innerhalb der Ohrplakode / otic Tasse neuralen sensorische zuständigen Domain (NSD) angegeben. Fate-Mapping - Daten belegen , dass die neuronale und sensorischen Linien verbunden sind und von der NSD entstehen in der antero-ventralen Bereich des otic Tasse befindet oder otocyst bei Mäusen und Huhn 4,5. Zunächst delaminieren Neuroblasten aus der NSD Vorrücken in den Neuronen des auditorischen-vestibulären Ganglion zu geben, die schließlich in Hör- und Gleichgewichts Ganglien gespalten. Diese Neuronen innervieren die sensorischen HCs des Innenohrs und Kerne im Hirnstamm. Die Zellen thbei der NSD bleiben werden gedacht, Anlass zu verschiedenen Sinnes Patches zu geben, einschließlich Gehörsinnesorgan, der mechano-Umwandlungs-sensorischen HCs und ihre zugehörigen SCs aus. In den beiden Küken und Maus, Hörorgan sensorischen Vorläuferzellzyklus verlassen und HC Differenzierung treten bei Steigungen entgegenwirkt. In Küken beginnt auditorischen Vorläuferzellzyklus - Ausgang um ~ E5 und schreitet von der Basis bis zur Spitze, und vom Zentrum zur Peripherie 6. Einen Tag später HC Differenzierung beginnt im Scheitel und schreitet zu der Basis 7. Das Studium der Entwicklung des Innenohres in Huhn bietet viele technische Vorteile als Funktionsmechanismen können mit relativer Leichtigkeit in ovo im Gegensatz zu manipulieren Embryonen in utero in anderen Modellsystemen untersucht werden, die komplexe Operationen erfordert. Das hier beschriebene Verfahren nutzt in ovo Mikro Elektroporation Gene von Interesse in der vermutlichen Basila Zielr Papille Bereich anterior-ventral in dem Ohrbläschen speziell bei E4. Elektroporation in ovo ist eine Technik , die gut etabliert ist und häufig verwendet 8-14. Das Prinzip der Elektroporation Technik basiert auf der Tatsache , dass Nukleotiden (zB Plasmid - DNA, synthetische DNA oder RNA - Oligonucleotide) sind negativ geladen. Die DNA wird in das interessierende Gewebe injiziert. Wenn ein elektrischer Strom über das Gewebe gelegt wird, öffnet sich der Strom transiente Poren in den Zellwänden und ermöglicht die Aufnahme der DNA, da die negativ geladenen DNA der positiven Elektrode fließt in Richtung (Anode). Für gain-of-function Experimente werden Gene von Interesse in Expressionsplasmide gewöhnlich subkloniert, die geeigneten Expressionskassetten für das grün fluoreszierende Protein (GFP) oder rot fluoreszierendes Protein (RFP) enthalten. Für loss-of-function Experimente plasmid-basierten dominant negative Repressor-Konstrukten oder RNAi oder Morpholino Konstrukte commonly verwendet 15,16. Zur Hemmung microRNA (miRNA) Funktion miRNAs Schwamm Konstrukte, die typischerweise Plasmid basiert, 17 verwendet werden. Die hier beschriebene Methode ermöglicht es, die molekularen Mechanismen zu untersuchen , die Proliferation und Differenzierung in Hörorgans steuern, wie die in ovo Mikro Elektroporationsverfahren kann leicht durch Zugabe von mit in ovo - Zellproliferationstest kombiniert werden EdU auf den gesamten Embryo. Die Elektroporation und die Zugabe von EdU bei E4 in der Ohrbläschen durchgeführt, die einen Tag vor dem Beginn der Zellzyklus-Ausgang in dem Hörorgan ist. Die Analyse des Hörorgan ist in der Regel durchgeführt, 18-96 Stunden nach der Elektroporation bei E5 (Beginn der sensorischen Vorläuferzellzyklus-Ausgang), E6 (Beginn der HC Differenzierung) und E7 (bei HC Differenzierung); und später bis ~ E9 (Ende HC Differenzierung). Dieses Elektroporation Verfahren des Gentransfers ist vergänglich etwa ~ 4 Tage dauert, because die Gene von Interesse nicht integrieren in das Genom, aber das Verfahren ist anwendbar für die Verwendung mit geeigneten Plasmid - DNA-basierten Expressionsvektoren, die die Fähigkeit tun haben , in das Genom, beispielsweise Tol2 vermittelten Gentransfer 11 zu integrieren. Mit dieser Methode robust GFP oder RFP Ausdruck dauert ~ 4 Tage in der Cochlea, nach welchem ​​Punkt die Signale verblassen, noch bieten eine reichliche Zeitfenster die komplizierte Entwicklung des Hörorgan zu studieren. Diese in - ovo - Gentransferverfahren ist neu und ermöglicht, speziell auf die vermutliche Hörorgan bei E4 Ziel, die für die Untersuchungen optimal ist, die im Hörorgan auf HC Entwicklung konzentrieren. Es ist eine gute Ergänzung zu alternative Verfahren, die bei viel jüngeren Entwicklungsstadien 11,12 oder im Vergleich zur Verwendung von in vitro basilar Papillen Explantatkulturen 18 elektroporieren.

<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>
		<tr height="21">
			<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>
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			<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>
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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>
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			<td height="21" style="height:21px;">Micropippette Puller for pulling needles</td>
			<td>Sutter Instrument Co.</td>
			<td>P-97</td>
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			<td height="21" style="height:21px;">2.5x2.5 mm Box Platinum Heating Filament</td>
			<td>Sutter Instrument Co.</td>
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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>
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			<td height="21" style="height:21px;">Hamilton Glass Syringe 100 &#956;l</td>
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			<td height="21" style="height:21px;">Mineral Oil (Heavy) for Hamilton Glass Syringe</td>
			<td>Fisher Scientific</td>
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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>
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			<td height="21" style="height:21px;">3 cc Disposable Syringes</td>
			<td>Becton Dickinson and Company (BD)</td>
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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>
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			<td height="21" style="height:21px;">One pair of 2 mm Platinum Electrodes</td>
			<td>Bulldog Bio. / Nepagene</td>
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			<td>Bulldog Bio. / Nepagene</td>
			<td>CUY580</td>
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</table>

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

Hühnerembryonen sind ideale Modellsysteme zur Untersuchung embryonaler Entwicklung als Manipulationen der Genfunktion kann mit relativer Leichtigkeit in ovo ausgeführt werden. Das Innenohr Gehörsinnesorgan ist für unsere Fähigkeit, kritisch zu hören. Es beherbergt eine hochspezialisierte Sinnesepithel, die von mechano-Umwandlungs-Haarzellen (HC-) und der umgebenden Glia artigen Stützzellen (SCs) besteht. Trotz struktureller Unterschiede in den Gehörorgane, sind die molekularen Mechanismen , die Entwicklung des Hörorgans Regulieren evolutionär konservierten zwischen Säugetieren und Aves In ovo Elektroporation frühen Stadien bei E1 weitgehend begrenzt ist -. E3. Aufgrund der relativ späte Entwicklung des Hörorgan bei E5, Manipulationen des Hörorgan durch in ovo Elektroporation Vergangenheit E3 sind aufgrund der fortgeschrittenen Entwicklung des Hühnerembryo schwer in späteren Phasen. Die hier vorgestellte Methode ist eine vorübergehende Gentransferverfahren für Gene von Interesse Targeting anStufe E4 - E4.5 im Hühner auditorischen Sinnesorgan über in - ovo - Mikro-Elektroporation zu entwickeln. Dieses Verfahren ist anwendbar für Verstärkungs- und loss-of-Funktionen mit herkömmlichen Plasmid - DNA-basierten Expressionsvektoren und kann durch Zugabe von EDU (5-ethinyl-2&#39;-desoxyuridin) zu den gesamten Embryo in der mit in ovo - Zellproliferationstest kombiniert werden Zeitpunkt der Elektroporation. Die Verwendung von grün oder rot fluoreszierende Protein (GFP bzw. RFP) Expressionsplasmide ermöglicht dem Experimentator, um schnell festzustellen, ob die Elektroporation erfolgreich den auditorischen Abschnitt des Entwicklungsinnenohr abgezielt. Bei diesem Verfahren wird Papier, repräsentative Beispiele für GFP elektroporiert Proben veranschaulicht; 96 Stunden nach der Elektroporation und des Hörorgans zur pro-sensorischen Bereich der GFP - Targeting wurde von RNA in situ - Hybridisierung bestätigt - Embryonen wurden 18 geerntet. Das Verfahren Papier stellt auch ein optimiertes Protokoll für die Verwendung des EdU Thymidinanalogon Zelle prolif zu analysierenration; ein Beispiel für eine EdU basierte Zellproliferationsassays, die Immunmarkierung kombiniert und klicken Sie auf EdU Chemie vorgesehen.

Bei diesem Verfahren wird DNA Papier Plasmid des grün fluoreszierenden Proteins aus (GFP) Expressionskassetten wurde in die Entwicklung von Hühner basilar Papille gezielte (BP) mit optimierten Parametern von 12 V und 4 Impulsen mit 100 msec Impulsdauer und Intervalle von 200 ms, ein Nachgeben ~ 50% Embryo-Überlebensrate und Effizienz von Plasmid-DNA in die BP-Targeting. Fluorescent Imaging von nativen GFP - Expression Embryonen in Entwicklungs zeigte diese Methode der Elektroporation ...

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

Gentransfer in das Huhn Hörorgan von<em&gt; In Ovo</em&gt; Micro-Elektroporation

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 Aufrufe.  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. 

Serie: 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.

Effizienten Gentransfer in Küken Netzhäute für primäre Zellkulturstudien: Eine<em&gt; Ex-ovo</em&gt; Elektroporation Ansatz

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

Forschung

Entwicklungsbiologie

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.

Kultur von embryonalen Maus Cochlear Explantate und Gentransfer durch Elektroporation

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.

Methoden für die genau zu lokalisieren Übertragung von Zellen oder DNA in frühen Postimplantations Maus Embryonen

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.

Pfropfen von Beads in die Entwicklung von Hühnerembryo-Glieder zu identifizieren Signaltransduktionswegen Beeinflussung der Genexpression

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).

Ein Roman<em&gt; Ex Ovo</em&gt; Banding-Technik intrakardialer Hämodynamik in einem embryonalen Hühner System ändern

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 im Hühnchen auditorischen Hirnstamm

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-vermittelten Verlust der Funktionsanalyse in zerebelläre Untermodul Zellen mit In Utero Elektroporation-basierte Gentransfer

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.

Dreidimensionale organotypischen Kulturen der vestibulären und akustische Sinnesorgane

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.

Verwendung von Organkulturen vom Trowell-Typ zur Untersuchung der Regulation von dentalen Stammzellen

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.

Isolierung von mesenchymalen Stammzellen des Fettgewebes von Ratten zur Differenzierung in insulinproduzierende Zellen

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