Name: culture of embryonic mouse cochlear explants and gene transfer by electroporation
Uploaded: 2015-01-12T14:45:00.000+00:00
Duration: 9 min 3 s
Description: Watch this Scientific Journal Video about Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation at JoVE.com

We would like to acknowledge Dr. Bradley Schulte for comments on this protocol. This work was supported by National Institutes of Health grant R00 (5R00DC010220). This project was performed in a renovated laboratory space supported by Grant C06RR014516.

HINWEIS: Alle Protokolle mit lebenden Tieren müssen überprüft und durch ein Institutional Animal Care und Verwenden Committee (IACUC) zugelassen worden sein und offiziell anerkannte Methoden für die Pflege und Verwendung von Labortieren folgen. Alle Sektionen sollte mit einer sterilen Technik auf eine saubere Sterilbank werden. Handschuhe und eine Maske, falls erwünscht, sollte bei diesem Verfahren getragen werden. 1. Präparation der embryonalen Maus-Ear- <ol><li> Einrichten für Corti-Organ Explantatkulturen: <ol><li> Sterilisieren der Laminarströmung Gewebekulturhaube durch Einschalten UV-Licht für ungefähr 30 min und desinfiziert die Oberfläche mit 70% igem Ethanol vor der Verwendung. Zusätzlich zu sterilisieren alle Dissektionsinstrumente einschließlich feiner Dissektion Werkzeuge und Sylgard-beschichtete Platten für Embryo Dissektion über Autoklaven oder durch Eintauchen in 70% Ethanol für mindestens 20 Minuten vor dem Gebrauch. Gießen Sie 70% Ethanol und lassen Sie die Speisen und Instrumente an der Luft trocknen in einem sauberen laminare Strömung clean Bank vor der Verwendung. </li><li> Vorbereitung steriler Hanks Balanced Salt Solution (HBSS) / (4- (2-Hydroxyethyl) -1-piperazinethansulfonsäure) (HEPES) Mischung durch Zugabe von 10% 10x HBSS und 0,5% HEPES und den pH-Wert auf etwa 7,2 und Filter sterilisieren endgültige Lösung und bei 4 ° C. Führen Sie alle Sektionen in gekühlt HBSS / HEPES-Lösung zu einer besseren Erhaltung der Gewebe während des gesamten Verfahrens. </li><li> Beschichtung von Glasbodenschalen mit Basalmembranmatrix durch Zugabe von 5 ml kaltem (4 ° C) sterile Dulbeccos Modified Eagle Medium (DMEM) zu einem 300 &amp; mgr; l Aliquot von Basalmembran-Matrix in einem sterilen 15 ml-Polypropylenröhrchen. Vortexen Inhalt und mit 150 ul der Basalmembranmatrix-DMEM Mischung auf die Mitte jeder Kulturschale, so dass es den in der Mitte der Schale gut vorliegenden gesamte Kultur umfasst. Decken Sie die Speisen und an einem 37 ° C Inkubator für mindestens 45 Minuten vor dem Gebrauch. </li><li> Gießen Sie 4-5 ml gekühlt HBSS umgree sterile Polystyrol-Petrischalen und verlassen in der Haube geschlossen ist. </li><li> Bereiten Kulturmedium in einem sterilen 15 ml-Polypropylenröhrchen durch Zugabe von 9 ml DMEM werden 100 ul N2 Ergänzungen, 10 g / ml Cipro und 1 ml FBS. </li></ol></li><li> Die Isolierung der Embryonen: HINWEIS: In diesem Verfahren zum Kultivieren von Cochlea-Explantate aus embryonalen Mäuse, Ernte Innenohren aus embryonalen Tag (E) 13 Schläfenbeine mit dem Tag nach der Befruchtung als E1 9 berücksichtigt. Das Protokoll kann auch mit embryonalen Maus inneren Ohren beginnend von E12 bis E18 verwendet werden. <ol><li> Euthanize eine zeitgesteuerte schwangeren Maus mit CO 2 und opfern Tier durch Genickbruch oder entsprechend zugelassenen Protokollen. Führen Sie die Euthanasie von Laminar-Flow-Gewebekultur Haube, um die Sterilität in der Gewebekulturraum zu erhalten. </li><li> Zeigen eingeschläfert Maus auf Papiertuch mit Bauch nach oben und desinfizieren Bauch mit 70% Ethanol. </li><li> Öffnen Sie die Bauchhöhle durch Greifen der Hautmit einer gebogenen Pinzette mit einer Hand und schnitt Epidermis und Muskeln entlang der Mittellinie mit einer Schere mit einer anderen Hand. Machen Sie zwei senkrechte Schnitte auf beiden Seiten und ziehen Sie den Uterus sorgfältig durch Anheben bilateralen Gebärmutterhörner mit einer Pinzette und entfernen Sie sie von unten Binde mit Gewebe Schere. </li><li> Setzen Sie den embryonalen Kette in einer Petrischale mit Kalt Dissektion Lösung (HBSS / HEPES-Gemisch) und Transfer zum Laminar-Flow-Sterilbank. </li><li> Embryonen vorsichtig aus Plazenta und legen Sie sie in einem der sterile Petrischalen mit Dissektion Lösung. Wenn die Lösung färbt sich blutig, verschieben Sie sie in eine neue Petrischale. HINWEIS: An dieser Stelle können Embryonen mit einem Theiler Inszenierung Führung statt. </li><li> Enthaupten Embryonen durch Kneifen im Halsbereich mit einem Paar von sauberen Pinzette oder Schere und legen die Köpfe in einer Petrischale mit frisch gekühlt Sezieren Lösung. </li></ol></li><li> Dissection der inneren Ohren: <ol><li> Ort one Embryokopf in einem sterilen Sylgard Schale mit kaltem Seziertisch </li><li> Lösung. Arbeiten unter einem Binokular mit einer Vergrößerung von ~ 1,6X, immobilisieren den Embryo Kopf, indem Minutien Stifte oder um näher an Augenbereich (1A). </li><li> Mit zwei Paar sterile Pinzette vorsichtig die Haut und die Öffnung des Schädels auf der Rückenseite entlang der Mittellinie (Abbildung 1B). Entfernen Gehirn von Schädelhöhle und Innenohren im Schläfenbeine befindet, kann in diesem Stadium (1C, D) identifiziert werden. Die Auskleidung des Blutgefäßes, die normalerweise um den Innenohren vorhanden ist, kann als Bezugspunkt für die Identifizierung des Innenohrs in dieser Phase (1D) verwendet werden. </li><li> Präparieren Sie die Innenohr von der zeitlichen Knochen, indem die Zange unterhalb des Gewebes und Isolierung des Innenohrs von der Schädelbasis und in ein neues Gericht, das kalt Sezieren Lösung (1E, F). </li></ol></li></ol> 2. Erzeugung von Corti-Organ Explantatkulturen HINWEIS: In diesem Stadium der Entwicklung wird das Gewebe Knorpel und kann mit einer Pinzette leicht präpariert werden. <ol><li> Richten Sie das Innenohr, so dass Bauchseite nach oben zeigt (Abbildung 2A) und Stabilisierung des Innenohrs durch leichtes Einsetzen des scharfen Ende Minutien Stifte durch die vestibulären Teil des Innenohrs (2B). </li><li> Mit ultrafeinen Zange aufgeschnitten das darüber liegende Knorpel durch einen Einschnitt mit einem Ende der Zange in der Nähe von dem ovalen Fenster und entfernen Sie vorsichtig den Knorpel aus der Hörschnecke (2C). Hinweis: Es ist wichtig, sicherzustellen, dass Zange nicht zu tief in den Knorpel einge als darüberliegenden Knorpel wird manchmal mit dem Schneckengang in diesem Fall ist es hilfreich, geschlossener Zange verwenden, um Knorpel von dem darunterliegenden Schneckengang frei durch Abkratzen unter der kondensierte sberfl des Knorpels. In diesem Stadium der Entwicklung, ist die Spirale der Cochlea nur drei Viertel einer Umdrehung in der Länge. </li><li> Als nächstes setzen die Sinnesepithel indem die Zange in dem bevorzugten Bereich der Schneckengang entweder der Basis oder an der Spitze sehr, und sanft herausziehen das Dach der Cochlea (2D). HINWEIS: Das Dach der Cochlea muss vollständig entfernt werden, da es die Sinnesepithel behindern die Analyse maskieren. HINWEIS: Dieses Verfahren muss vorsichtig erfolgen, da es einfach ist, die Sinnesepithel reißen werden. </li><li> Als letzter Schritt, vorsichtig die zugrunde liegenden Bindegewebe von der freiliegenden Sinnesepithel so dass die Basis der Cochlea-Explantat gleichmäßig flach. </li><li> Isolieren Sie die seziert Cochlea von der vestibulären Teil des Innenohrs mit einer Pinzette. Dies kann vor oder nach dem Schritt 2.4 (2E) durchgeführt werden. </li><li> Übertragen Sie die seziert Cochlea Sinnesepithel in einen Keller membrane Matrix-beschichteten Kultur auch mit einem 1,5 mm sterile scooper (2F). </li><li> Richten Sie die Cochlea-Explantat mit der Lumenoberfläche des Epithels nach oben ein und saugen Sie den Basalmembranmatrix-DMEM-Lösung sorgfältig zu glätten das Gewebe und ersetzen Sie es mit 150 ml frisches Kulturmedium durch sanftes Zugabe in die Schale. Ist darauf zu achten, um sicherzustellen, dass jedes Explantat haftet gut auf der beschichteten Glasdeckglas und wird nicht in das Kulturmedium schwimm werden. Stellen Sie sicher, dass die Spitze der Zange wird darauf hingewiesen, während der Anbringung des Glases auch um eine Beschädigung der Cochlea Explantation. </li><li> Die Kulturschale vorsichtig Führung in eine sterile 150 mm Kulturschale und in eine Gewebekultur-Inkubator bei 37 ° C mit 5% CO 2 für eine gewünschte Zeitdauer, in der Regel 3-6 Tage in vitro (DIV). Nach 1 DIV, untersuchen Sie die Kulturen unter einem Binokular, um sicherzustellen, dass die Cochlea-Explantat wurde auch in die Kulturschale eingehalten werden. </li><li> Nach dem incubation in vitro für die gewünschten Zeitdauern werden die Kulturen für die Immunhistochemie verarbeitet. HINWEIS: Die Explantatkulturen Regel werden 6 DIV das entspricht Entwicklungsstufe, P0 inkubiert. Nach Inkubation in vitro können Kulturen für Downstream-Anwendungen wie die Immunhistochemie (3B) verarbeitet werden, RT-PCR, in situ-Hybridisierung und / oder Western Blot. </li></ol> 3. Elektroporation vermittelte Gentransfer <ol><li> Die Einrichtung für die Elektroporation: <ol><li> Sterilisieren Sie einen 100 mm Sylgard-beschichtete Glasschüssel durch Autoklavieren oder Eintauchen in 70% Ethanol für etwa 20 Minuten und an der Luft trocknen in einer Sterilbank vor der Verwendung. </li><li> Bereiten DNA aus einem Expressionsvektor der Wahl mit geeigneten Maxi- oder Midi-Prep-Kits. Die in diesem Protokoll verwendeten Expressionsvektoren sind pIRES2-Atoh1.EGFP und pCLIG-NeuroD1.EGFP. Die Endkonzentration der DNA sollte mindestens 1 ug / ul in sterilem DNA sein /RNase-freies Wasser. </li></ol></li><li> Elektroporation DNA in embryonale Cochlea Explantate: <ol><li> Fügen Sie 10 ul der Plasmid-DNA-Lösung auf eine frische 100 mm Sylgard beschichtete Schale und übertragen eine komplett zerlegt Cochlea Explantation (aus Schritt 2.5 erhalten) in die DNA-Lösung. </li><li> Mit Lumenoberfläche des Epithels nach oben, kippen Sie das Cochlea leicht, so dass es senkrecht zur Ebene der Schale. </li><li> Die Elektrode Schaufeln auf beiden Seiten der Cochlea mit negativen Paddel (Kathode) in Richtung des sensorischen Epithels und der positiven Paddel (Anode), die benachbart zu der Basis der Cochlea. Hinweis: Da die DNA negativ geladen ist, von negativ nach positiv Elektrode und durch die Plazierung einer Kathode Ende der Sonde angrenzend an das Riechepithel wandert es; die DNA wird vor allem durch die Oberfläche des Sinnesepithel gehen. </li><li> Mit Elektroporator liefern 9-10 Pulse von 24 mV, 30 ms Pulsdauer mit dem Fußschalter auf T-er Elektroporator, und fügen Sie 100 ul von warmem Kulturmedium, um die elektroporierten Cochlea. </li><li> Die Schritte 3.2.1-4 für alle Cochlea Explantate und DNA der Gene von Interesse und übertragen die elektroporierten Cochleae in die Basalmembran-Matrix-beschichtete Schale zum Plattieren (Schritt 2.7). Kultur ganze elektroporiert Cochleae in einem 37 ° C, 5% CO 2 befeuchteten Inkubator für gewünschte Zeitdauern, die dann für Immunzytochemie verarbeitet werden. HINWEIS: Die Schnecke aus einem ganzen Wurf von CD1 Maus (ungefähr 8-12 Welpen) isoliert werden, mikrodissezierten, elektroporiert und in &lt;4 Stunden überzogen. </li></ol></li></ol> 4. Analyse der Cochlear Explantatkulturen HINWEIS: Die Kulturen werden in der Regel für 6 DIV, die äquivalent zu der Entwicklungsstufe, P0 ist, inkubiert. Nach Inkubation in vitro werden die Kulturen fixiert und für Immunzytochemie verarbeitet. <ol><li> Nach gewünschte Länge incubation Entfernen Kulturmedium schnell und gründlich die Cochlea Explantat in phosphatgepufferter Salzlösung (PBS) und Inkubation in 4% Paraformaldehyd (in PBS) für 15 min bei Raumtemperatur. </li><li> 15 min Waschen Sie die Fixiermittel durch Zugabe von PBS und spülen in PBS dreimal. </li><li> Permeabilisieren Verwendung von PBS-T (PBS + 0,5% Tween) für 30 min vor dem Blockieren mit PBS-T, enthaltend 10% Ziegenserum. </li><li> Inkubiere in primären Antikörperlösung, die Primärantikörpers in PBS-T mit 1% Ziegenserum über Nacht bei 4 ° C. </li><li> Dreimal Waschen Sie die Cochlea bei Raumtemperatur mit PBS-T jede 15 Minuten, bevor Sie entsprechende Alexa-konjugierten Sekundärantikörper. </li><li> Da der Cochlea Explantat auf Glasdeckgläschen kultiviert sie können einzeln durch Isolierung des Deckglases von der Schale und die Montage auf einer Folie verarbeitet werden. Dies kann durch Eintauchen der Kulturschale in OS30 Lösungsmittel für mindestens 1 Stunde bei Raumtemperatur erreicht werden. Mit dem sterilen Rasierklinge, nehmen Sie den Deckglas aus der dish und montieren auf einer Folie und sichtbar unter einem Fluoreszenzmikroskop. </li></ol>

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No conflicts of interest declared.

Alle Zellen innerhalb des membranösen Labyrinth des Maus Innenohres einschließlich sensorischer, nonsensory und die Spiralganglienneuronen sind alle aus placodally abgeleiteten otocyst benachbart zu dem Hinterhirn des Ektoderms rund E8 10-14 abgeleitet ist. Bei E11 ventralen Bereich des otocyst erstreckt, um den Schneckengang zu bilden, und die Entwicklung geht weiter, eine Gruppe von Epithelzellen in der Cochlea als auch in anderen Bereichen des otocyst, werden als prosensory Patches, anschließend Hieraus...

The mammalian organ of Corti is comprised of a mosaic of specialized cell types, including two types of mechanosensory hair cells as well as at least four types of nonsensory supporting cells making it an ideal model system to study normal cellular processes like proliferation, fate specification, differentiation and patterning. In addition, the normal development of these different cell types is essential for normal hearing function. Hence, it is crucial to understand the factors, both molecular and cellular, that regulate their development. However, the small size of the mouse cochlea as well as its inaccessibility poses a particular challenge for gene expression studies. Moreover, most of the cell fate specification and patterning events occur during embryonic time periods and are mostly completed before birth. Therefore, identification and characterization of signaling events during embryonic time periods is essential to gain insight into the molecular basis of cochlear morphogenesis. 
Here, we demonstrate a method to culture intact cochlea in vitro from embryonic mouse inner ears. The rationale behind the use of this technique is that cultured cochleae maintain their molecular and morphological characteristics thereby providing a valuable model for investigating potential candidate genes and exploring the mechanisms involved in cochlear morphogenesis. Although transgenic mice can be used for gene expression studies, an in vitro system is often needed for monitoring specific gene functions. Moreover, cochlear cultures can be established from transgenic mouse embryos so that their in vitro development and response to various soluble factors and antagonists can be studied. Although embryos at day 13 (E13) are used in this protocol, cultures from E12 or E14 to early postnatal inner ears can give similar results. 
We also present a gene transfer technique in cultured embryonic cochlear explants using square wave electroporation. Following the isolation of the cochlear explants, electroporation can be used to express DNA plasmids of gene(s) of interest in individual cells within the cochlear duct. This technique serves as a complementary approach to studies utilizing transgenic mice to gain insight into the molecular pathways underlying cellular phenotype. Using this method of gene transfer, a variety of epithelial cell types within the embryonic cochlea are transfected, thereby enabling loss-and gain-of-function analyses at the single-cell level. In addition, electrophysiological studies can also be performed in cochlear explant cultures8. This method of in vitro electroporation is relatively simple and straightforward, combined with minimal damage to the tissue, has resulted in a rapid expansion of this technique.

<table><tbody><tr><td>HBSS</td><td>Gibco</td><td>14065-056</td><td></td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Medium</td><td>Gibco</td><td>12430-054</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>10082</td><td></td></tr><tr><td>N-2 Supplement (100x)</td><td>Gibco</td><td>17502-048</td><td></td></tr><tr><td>Ciprofloxacin hydrochloride</td><td>Cellgro</td><td>61-277-RF</td><td></td></tr><tr><td>Glass Dish 60 mm</td><td>Kimble Chase</td><td>23062-6015/23064-6015</td><td></td></tr><tr><td>Glass Dish 100 mm</td><td>Kimble Chase</td><td>23064-10015/23062-10015</td><td></td></tr><tr><td>Minutien pins</td><td>Fine Science Tools</td><td>26002-15</td><td></td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools</td><td>11251-10</td><td></td></tr><tr><td>Pulse generator</td><td>Protech International Inc</td><td>CUY21Vivo-SQ</td><td></td></tr><tr><td>Glass bottom culture dishes</td><td>MatTek</td><td>P35G-0-10-C</td><td></td></tr><tr><td>Matrigel matrix</td><td>BD Biosciences</td><td>356237</td><td></td></tr><tr><td>Culture dish, 60 x 15 mm</td><td>Becton Dickinson</td><td>353037</td><td></td></tr><tr><td>Tissue culture dishes</td><td>Greiner Bio-one</td><td>639160</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>OS-30</td><td>Dow Corning</td><td>4021768</td><td></td></tr><tr><td>Fluoromount</td><td>Southern Biotech</td><td>0100-01</td><td></td></tr><tr><td>Conical tubes, 15 ml</td><td>Greiner Bio-one</td><td>188261</td><td></td></tr><tr><td>Myosin 6</td><td>Proteus Biosciences Inc</td><td>25-6791</td><td></td></tr><tr><td>Myosin 7a</td><td>Proteus Biosciences Inc</td><td>25-6790</td><td></td></tr><tr><td>TuJ1</td><td>Sigma</td><td>T2200</td><td></td></tr></tbody></table>

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 describe a method to isolate cochlea from embryonic inner ears and micro-dissect to expose the sensory epithelium. Once dissected, it may be plated and cultured as an intact cochlear duct (Figure 3) and analyzed by immunohistochemistry. The cultured cochlear explants provide a useful assay to examine the effect of variety of soluble factors and pharmacological drugs on cochlear development. Following dissection of cochlear explants, electroporation technique can be used to misexpress genes of interest...

Watch this Scientific Journal Video about Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation at JoVE.com

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

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 ...

culture-embryonic-mouse-cochlear-explants-gene-transfer

Research

JoVE Journal

Developmental Biology

12.8K Aufrufe.  Medical University of South Carolina, College of Medicine. 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. 

Serie: Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

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, ...

Forschung

Entwicklungsbiologie

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

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

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

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.

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

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

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) has been shown to be a useful approach to local drug delivery into the inner ear. The goal of this article is to describe, in detail, the surgical techniques involved in canalostomy in both adult and neonatal mice. As indicated by fast-green dye and adeno-associated virus serotype 8 with the green fluorescent protein gene, the canalostomy facilitated broad distribution of injected reagents in the cochlea and vestibular end-organs with minimal damage to hearing and vestibular function. The surgery was successfully implemented in both adult and neonatal mice; indeed, multiple surgeries could be performed if required. In conclusion, canalostomy is an effective and safe approach to drug delivery into the inner ears of adult and neonatal mice and may be used to treat human inner ear diseases in the future.

Here we describe canalostomy procedure which allows local drug delivery into the inner ears of adult and neonatal mice through the semicircular canal with minimum damage to hearing and vestibular function. This method can be used to inoculate viral vectors, pharmaceuticals, and small molecules into the mouse inner ear.

Canalostomy als ein chirurgisches Vorgehen zu Local Drug Delivery in das innere Ohr von Erwachsenen und Neugeborenen Mäusen

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

Genetik

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

In-vivo-Elektroporation von Entwicklung Maus Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Neurowissenschaften

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 ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

Primäre Kultur und Plasmid Elektroporation der Murine Corti-Organ.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Biologie

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

Langfristige Zeitraffer-Imaging von Maus Cochlear Explantate

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

An enhanced platform for whole-embryo culture allows continuous and robust ex utero development of postimplantation mouse embryos for up to six days, from pregastrulation stages until advanced organogenesis. In this protocol, we detail the standard procedure for successful embryo culture using static plates and rotating bottle systems.

Ex Utero Kultur von Mausembryonen von der Prägastrulation bis zur fortgeschrittenen Organogenese

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. ...

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 ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

Neonatal Murine Cochlear Explant Technik als<em&gt; In vitro</em&gt; Screening-Tool in der Hörforschung

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Kultur von embryonalen Maus Cochlear Explantate und Gentransfer durch Elektroporation

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