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>

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

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 ...

Forschung

Entwicklungsbiologie

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

In Ovo Electroporations von HH Stage 10 Hühnerembryonen

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

Biologie

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 ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

Gentransfer an sich entwickelnden Maus Innenohr<em&gt; In Vivo</em&gt; Die Elektroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Neurowissenschaften

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

In ovo Elektroporation in Mittelhirn-Küken für Studien über Genfunktion in dopaminergen Neuronen Entwicklung

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

In ovo Expression von microRNA in ventralen Mittelhirn-Küken

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

In Ovo und Ex Ovo Methoden zur Untersuchung der Entwicklung des Vogelinnenohrs

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons1, which has been difficult with the conventional method of injection and electroporation at E1.52.

In Ovo Electroporation in Embryonic Chick Retina

The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.

In ovo Elektroporation in embryonalen Hühnchenretina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

Gentransfer in Hühnerembryonen durch Ältere<em&gt; Ex ovo</em&gt; Die Elektroporation

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Fragile X mental retardation protein (FMRP) is an mRNA-binding protein that regulates local protein translation. FMRP loss or dysfunction leads to aberrant neuronal and synaptic activities in fragile X syndrome (FXS), which is characterized by intellectual disability, sensory abnormalities, and social communication problems. Studies of FMRP function and FXS pathogenesis have primarily been conducted with Fmr1 (the gene encoding FMRP) knockout in transgenic animals. Here we report an in vivo method for determining the cell-autonomous function of FMRP during the period of circuit assembly and synaptic formation using chicken embryos. This method employs stage-, site-, and direction-specific electroporation of a drug-inducible vector system containing Fmr1 small hairpin RNA (shRNA) and an EGFP reporter. With this method, we achieved selective FMRP knockdown in the auditory ganglion (AG) and in one of its brainstem targets, the nucleus magnocellularis (NM), thus providing a component-specific manipulation within the AG-NM circuit. Additionally, the mosaic pattern of the transfection allows within-animal controls and neighboring neuron/fiber comparisons for enhanced reliability and sensitivity in data analyzing. The inducible vector system provides temporal control of gene editing onset to minimize accumulating developmental effects. The combination of these strategies provides an innovative tool to dissect the cell-autonomous function of FMRP in synaptic and circuit development.

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Using in ovo electroporation, we devised a method to selectively transfect the auditory inner ear and cochlear nucleus in chicken embryos to achieve a cell-group-specific knockdown of fragile X mental retardation protein during discrete periods of circuit assembly.

Sezieren der zellautonomen Funktion des fragilen X-mentalen Retardierungsproteins in einem auditorischen Schaltkreis durch In-Ovo-Elektroporation

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