Wir bedanken uns bei Humana Press für die Erlaubnis, die Mikroinjektion Pipette Herstellung Figur, die ursprünglich auf Seite 130 der Verweis 11 erschienen zu veröffentlichen; Larry Dlugas und Steven Wong, OHSU Abteilung Pädagogische Kommunikation, für die Videografie; Larry Dlugas für Video-Design und-Bearbeitung; Adam M. O &#39;Quinn, Senior Designer, Trion / Envirco für die Gestaltung unserer kundenspezifischen horizontale Laminarströmungshaube und Les Goldsmith für die Bereitstellung der technischen schematische; Monterroso Victor, MW, MS, PhD und Tom Chatkupt, DVM, OHSU Abteilung für Vergleichende Medizin, zur Orientierung mit unseren Tierpflege-Protokoll, chirurgische Techniken, und eine prophylaktische Therapie Analgesie; Marcel Perret-Gentil, DVM, MS, für die gemeinsame Nutzung seiner Handzettel auf tierärztliche Nahttechniken; Edward Porsov, MS, für die Gestaltung unserer Adobe Premiere Pro Video-Mikroskopie Computer-Arbeitsplatz, und Leah und Weiß Jonas Hinckley von LNS Captioning (Portland, OR). Diese Arbeit wurde durch Zuschüsse aus dem National Institute on Taubheit und anderen, unterstütztr Communication Disorders: DC-und DC-008595 R01 R01 008.595-04S2 (JB) und P30 DC005983 (Oregon Hearing Research Center Core-Grant, Peter Gillespie, Principal Investigator).

1. Ventralen Laparotomie <ol><li> Anesthetize einen Damm, dessen Embryonen sind an embryonalen Tag 11,5 (E11.5; Uhr am Tag ein Vaginalpfropf erkannt wird ist Tag 0,5 der Embryonalentwicklung) durch intraperitoneale Injektion von Natrium-Pentobarbital Narkose-Lösung (7,5 ul pro Gramm Körpergewicht). Arbeiten Anästhesielösung: 180 ul 50 mg / ml Pentobarbital-Natrium-Lösung; 100 L absolutem Ethanol; 320 ul von 65 mg / ml wässrige Magnesiumsulfat (moduliert Uterustonus) und 400 ul Propylenglykol (Fahrzeug mischbar mit wässrigen und organischen Komponenten). </li><li> Beurteilen Sie die Vollständigkeit der Narkose durch die Durchführung Schmerzreize Tests: Squeeze Pfote; Schwanz Prise; und Blink-Reaktion auf die Wange und Tasthaare Touch. Bewerben sterile Augensalbe auf die Hornhaut. </li><li> Rasieren Sie die Bauch-Fell auf dem suprapubischen Bereich um den Brustkorb mit einer Schere und einem feinen Sägeblatt (Oster # 40 Klinge). Desinfizieren Sie den Bauch mit 70% Ethanol, 10% Povidon-Iod (Betadine), eineRunde 70% Ethanol, sequentiell. Bewegen Sie die Maus Bauch nach unten auf einem sterilen Tuch und dann auf einem Heizkissen oder warme Platte (37 ° C) für 2-5 Minuten eingestellt. </li><li> Inzision der Bauchhaut in der ventralen Mittellinie mit Ball-bestückte Schere. Verlängern Sie die Inzision für 10-14 mm. Identifizieren Sie die Linea alba, eine avaskuläre, weiß Bindegewebe Band entlang der ventralen Mittellinie der Bauchwand. Inzision der Linea alba mit Kugel gekippt Schere und erweitern den Schnitt 10-14 mm. Unmittelbar bewässern den Bauch mit vorgewärmtem (37 ° C) Ringer-Laktat-Lösung. </li><li> Externalisieren die beiden Hörner des Uterus mit Ring Zange ohne übermäßigen Druck auf den Nidationsstellen. Spülen Sie die ausgelagerten Uterushörner mit vorgewärmtem, Ringer-Laktat-Lösung. </li></ol> 2. Transuterine Mikroinjektion <ol><li> Fertigen Sie ein dickwandiges, Borosilikatglas Kapillare Mikroinjektion Pipette mit den folgenden Merkmalen: 12-16 μm Außendurchmesser und eine 20-Grad-Schräge. Auf einer Sutter P-97 Pipette Abzieher mit kleinen Kasten Filament, verwenden Sie das folgende Programm: Wärme = Rampe Test plus 3 Einheiten; Druck = 200; ziehen = 0; Geschwindigkeit = 46; Zeit = 100). Mit dem Sutter BV-10 Beveler, verwenden Sie den 104C (Gold) Schleifscheibe mit großem Durchmesser für Pipetten. Resuspendieren Expressionsplasmid bei 3-4 g / ul in Calcium-freier phosphatgepufferter Salzlösung (pH 7,2-7,4). In kristallinen schnell grün zu dem konzentrierten DNA, vorsichtig vortexen für 30 Sekunden, und Spin bei 10.000 g für 10 Sekunden. Die minimale Menge an schnell grün erforderlich ist, um visuell verfolgen die Wirksamkeit der Injektion wird empirisch bestimmt. Verfüllen die abgeschrägte Pipette mit dem DNA / schnell grüne Lösung. Verbinden des geladenen Mikroinjektion Pipette auf die Pipettenhalter der Injektor (Picospritzer mit&gt; 99% reinem Stickstoff als Quellengas). </li><li> Durchleuchten die Gebärmutter mit geringer Intensität, Halogenlicht, um den Embryo in seinem Implantationsstelle zu visualisieren. Identifizieren Sie den BEATing Herz, Gehirn Vesikel, Gliedmaßenknospen, im Entstehen begriffenen 4. Ventrikel des Hinterhirn und Auge. Spülen des Uterus alle 2 Minuten mit vorgewärmtem, Ringer-Laktat-Lösung, um die Hydratisierung zu halten. Drücken Sie vorsichtig auf die Gebärmutter, um den Embryo neu zu orientieren und Identifizierung der anatomischen Landmarken oben erwähnt. </li><li> Orient der Embryo, um den primären Kopf Vene, deren vordere und hintere Äste flankieren den mesenchymalen Gebiet, in dem die otocyst wohnt identifizieren. Die otocyst selbst kann nicht durch Durchleuchtung des Uterus werden. Die otocyst in der Mitte zwischen den vorderen und hinteren Äste der primären Kopf Vene, die, zusammen mit dem Hauptstamm der Vene, bilden die Form der Pfosten oder Torpfosten auf einem American-Football-Feld befindet. Die otocyst in der Mitte zwischen den Pfosten. </li><li> Legen Sie die Injektionspipette durch die Gebärmutter in einer Bahn im Einklang mit dem mutmaßlichen Ort der otocyst. Impuls die Mikroinjektorkopfs einmal nach dem Durchlaufen der uteruns zur Visualisierung der Tracer-Farbstoff und die ungefähre die Position der Pipettenspitze. Schieben Sie die Pipette unter Mikrometer-Steuerung und Puls wieder in die Tiefe zu beurteilen. Weiter voranzutreiben Sie die Pipette in die seitliche Kopf Mesenchym und Puls wiederholt. Erfolgreiche otocyst Targeting wird zeigen, die sich verjüngende Form des Ductus endolymphaticus dorsal und der Jakobsmuschel-Form der Apsis. Lassen Sie den Druck auf die Gebärmutter, entfernen Sie die Pipette aus dem Embryo / Gebärmutter in einer Bewegung, und sofort bewässern die Gebärmutter mit vorgewärmtem, Ringer-Laktat-Lösung. </li></ol> 3. In vivo Elektroporation <ol><li> Spülen Sie die Gebärmutter mit Ringer-Laktat-Lösung. Frisch aufgetragen Ringer-Laktat-Lösung ist notwendig, um elektrisch zu koppeln, die Paddel-Stil Elektroden in die Gebärmutter. Befeuchten Sie die Wolfram-Oberflächen der Elektroden mit Ringer-Laktat. Zentrum des eingespritzten otocyst in dem Weg der Elektroden. Sanft komprimieren die Gebärmutter mit der Elektroporation paddles. Die Kathode ist in Kontakt mit der Uteruswand quer zur injiziert otocyst und die Anode in Kontakt mit der Uteruswand benachbart zu der injizierten otocyst. Lösen Sie eine Rechteckimpuls Zug mit dem Fußpedal-Schalter auf der Elektroporator. Die Elektroporation Parameter sind: 5, 50 ms Pulse bei 43 Volt pro Puls und einer 950 ms Zwischenpulsverzögerung. Unmittelbar bewässern die Gebärmutter nach der Impulsfolge wird ausgeliefert. Notieren Sie die aktuelle Lieferung an das Gewebe: 60-100 mAmps ist ausreichend, um epithelialen Vorläuferzellen otic transfizieren. Injizieren und elektroporieren 4-6, E11.5 Embryonen pro Muttertier. </li><li> Durchführen einer zweiten, unabhängigen transuterine Mikroinjektion von wässrigen fluoreszierenden Dextran (Alexa Fluor 488, wenn der Ausdruck Plasmid codiert ein rot fluoreszierendes Protein oder Alex Fluor 594, wenn das Expressionsplasmid kodiert für ein grün fluoreszierendes Protein) in der 4. Ventrikels dieser Embryonen, deren innere Ohren genau gespritzt und mindestens 60 mAmps von Strom pro Puls war deangeliefert bei der Elektroporation. Das fluoreszierende Dextran wird bei der Geburt nachweisbar im Hinterhirn und ermöglichen die Auswahl der Jungtiere, deren innere Ohren wurden während der Embryogenese manipuliert (siehe 3.5). </li><li> Spülen Sie die Uterushörner mit Ringer-Laktat. Setzen Sie die Uterushörner in die Bauchhöhle. Spülen Sie die Gebärmutterhöhle mit 2-4 ml vorgewärmtem, Ringer-Laktat-Lösung und damit der Überlauf abfließen aus dem Inzisionsstelle auf den sterilen Tuch. Ersetzen Sie die Drapierungen bei trockenen, sterilen Material. Nähen Sie die Bauchdecke mit einer nicht schneidenden Nadel und einem resorbierbaren Nahtmaterial 6-0. Wir bevorzugen eine Vorstich, Sperren jede zweite Masche, sowohl für die Bauchdecke und die Haut. </li><li> Trocknen Sie die Dämme &#39;Fell und zu verwalten, eine nicht-steroidale Entzündungshemmer wie Meloxicam durch subkutane Injektion. Bringen Sie den Damm auf die vorgewärmten Erholung Käfig auf einem sterilen Tuch. Überwachen und Aufzeichnen der Dämme &#39;Atmung, Inzisionsstelle Durchgängigkeit und Vagina für blutigen Ausfluss. Blutungen sind rawieder, aber wenn vorhanden und unvermindert, einschläfern den Damm, während sie noch unter Narkose. Notieren Sie sich die Zeit, sie wieder zu sich und versucht, ambulate. Bringen Sie den Damm auf die Maus Kolonie, als sie Anzeichen für Essen und Trinken zeigt, und hat damit begonnen, Nest zu bauen. Normalerweise passiert dies innerhalb von 12 Stunden. </li><li> Bei der Geburt (postnatalen Tag 0), setzen Sie das Flash-Hinterhirn Bereich jeder Welpe im Wurf, den fluoreszierenden Dextran mit einem stereofluorescence Binokular mit einem GFP-oder Texas Red-Filter erkennt nach Bedarf. Zurück zur säugenden Mutter nur jene Welpen, die Rautenhirn Kennzeichnung anzuzeigen. </li></ol> 4. Repräsentative Ergebnisse <img src="/files/ftp_upload/3653/3653fig1.jpg" alt="1" /> Abbildung 1. Elektroporation vermittelte Gentransfer in die Entwicklung von Cochlea. Die E11.5 otocyst wurde mit einem Expressionsplasmids Enhanced Green Fluorescent Protein (EGFP) und elektroporierten injiziert (Puls train-Parameter: fünf, 43-Volt-Impulsen bei 50 ms / 950 ms Puls-und Puls-Verzögerung). A) ein Vertreter des Innenohrs von einer postnatalen Tag 6 (P6), deren Welpen otocyst wurde injiziert und bei E11.5 elektroporiert demonstrieren EGFP-Expression von der Basis durch die Mitte Windung der Cochlea. Die laterale Wand der Cochlea wurde aus der Mitte und der Spitze wiederum nur entfernt. E11.5 Vorfahren, die Anlass zu der Spitze wurden nicht transfiziert. B) Immunfärbung Whole mount in der Cochlea (A) mit dem Haar-Zell-Marker, zeigt Myosin 7a (MYO7A), dass EGFP-Expression folgt die Flugbahn des Corti-Organ und wird grob an die Haarzelle tragenden Sinnesepithel lokalisiert. C) einem Vertreter des Innenohrs von einem Embryo, dessen E18.5 otocyst wurde injiziert und bei E11.5 elektroporiert. Die Laser-Projektion zeigt konfokale EGFP-Expression in MYO7A-positiven sensorischen Haarzellen. D) konfokalen Laser-Projektion des Cochlea-Sinnesepithel aus dem E18.5 Corti-Organ anzeigt EGFP expression in der inneren Haarzellen (IHC), äußeren Haarzellen (OHC), innere phalangial Zellen (IPC), Pfeiler-Zellen (PC) und Deiters &#39;Zellen (DC). Der Balken in (B) gilt für (A).

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Gentransfer in sich entwickelnden Maus Innenohr: Das Innenohr der Maus entwickelt sich aus der Ohrplakode während der ersten Woche der Einnistung Entwicklung 12,13. Von embryonalen Tag 9,5 (E9.5) hat die Plakode eingestülpt und verwandelte sich in einem mit Flüssigkeit gefüllten Vesikel genannt otocyst 2. Otic Vorstufen in der Blase führen zu den sensorischen und nichtsinnlichen Zellen innerhalb des reifen Innenohr sowie die Neuronen, die mechanisch empfindlichen Haarzellen in...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Micro Sterilisieren Gehäuse </td><td> Roboz </td><td> RS-9900a </td><td> 8X8.5X1.25 Zoll </td></tr><tr><td> Kugel-bestückte Schere </td><td> Fine Science Tools </td><td> 14109-09 </td><td></td></tr><tr><td> Ring Zange </td><td> Fine Science Tools </td><td> 11106-09 </td><td> 4,8 mm ID/6mm OD </td></tr><tr><td> Adson Pinzette </td><td> Fine Science Tools </td><td> 11027-12 </td><td></td></tr><tr><td> Nadeltreiber </td><td> Fine Science Tools </td><td> 12502-12 </td><td></td></tr><tr><td> Allergie Spritzentablett </td><td> Becton Dickison </td><td> 305536 </td><td></td></tr><tr><td> Suture 6-0 </td><td> Syneture </td><td> GL-889 </td><td> 0,7 metrischen Magen-Darm-Naht </td></tr><tr><td> Ringer-Lactat-Injektion USP </td><td> Baxter </td><td> 2B2323 </td><td></td></tr><tr><td> Schnell grün </td><td> Sigma Aldrich </td><td> F7258 </td><td></td></tr><tr><td> Borosilikatglas Kapillare </td><td> Harvard Apparatus </td><td> 30-0053 </td><td></td></tr><tr><td> Nembutal Natriumlösung </td><td> OVATION Pharmaceuticals Inc. </td><td> NDC 67386-501-52 </td><td></td></tr><tr><td> MgSO4.7H2O </td><td> Fisher Scientific </td><td> M63-500 </td><td></td></tr><tr><td> Propylenglykol </td><td> Fisher Scientific </td><td> P355-1 </td><td></td></tr><tr><td> Ethanol </td><td> Sigma Aldrich </td><td> E7023-500 </td><td></td></tr><tr><td> Meloxicam </td><td> Boehringer Ingeheim </td><td> NADA 141-219 </td><td></td></tr><tr><td> Feinpipettenziehvorrichtung </td><td> Sutter Instruments </td><td> P-97 </td><td> FB255B Box Filament; konsultieren Pipette Kochbuch von Sutter Instrumente </td></tr><tr><td> Mikroelektroden Beveler </td><td> Sutter Instruments </td><td> BV-10 </td><td> 104C Abschrägung Festplatte für große Pipetten, konsultieren Sie in der Bedienungsanleitung Anfasen Theorie </td></tr><tr><td> Mikropipette Halter </td><td> Warner Instruments </td><td> MP-S15T </td><td> Für 1,5 mm Außendurchmesser Pipette und weiblichen Druckanschluss für Picospritzer Schlauch. </td></tr><tr><td> Pinzetten-Stil Elektrode </td><td> Protech International Inc. </td><td> CUY650P5 </td><td> 5 mm Außendurchmesser </td></tr><tr><td> Square Wave Electroporator </td><td> Protech International Inc. </td><td> CUY21EDIT </td><td> Footpedal empfohlenen </td></tr><tr><td> PICOSPRITZER III </td><td> Parker Hannifin </td><td> 051-0500-900 </td><td> Footpedal empfohlenen </td></tr><tr><td> Manuelle Steuerung Mikromanipulator </td><td> Harvard Apparatus </td><td> 640056 </td><td></td></tr><tr><td> Horizontale Laminar-Flow Sterilbank </td><td> Envirco </td><td></td><td> Kundenspezifische Modifikationen an LF 630-10554. Siehe ergänzende Informationen für schematische Kapuze. </td></tr><tr><td> Leica stereofluorescence Sezieren microcope mit Lumencor Sola Light Engine </td><td> Bartels und Stout und Lumencor </td><td> MZ10F mit Lumencor Sola Light Engine </td><td> Footpedals die MZ10F zu konzentrieren und die Sola Light Engine auslösen werden empfohlen </td></tr><tr><td> Alexa Fluor 594 Dextran </td><td> Invitrogen </td><td> D22913 </td><td> 10mg/ml, wässrigen </td></tr><tr><td> Alexa Fluor 488 Dextran </td><td> Invitrogen </td><td> D22910 </td><td> 10mg/ml, wässrigen </td></tr><tr><td> Enviro-dri </td><td> Shepherd Sonstige PapERS </td><td></td><td> <a href="http://www.ssponline.com" target="_blank">www.ssponline.com</a> </td></tr></tbody></table>

gene transfer to the developing mouse inner ear by in vivo electroporation

Die Säugetier-Innenohr hat 6 verschiedene Sinnesepithelien: 3 Cristae in den Ampullen der Bogengänge; Maculae im Utriculus und Sacculus, und das Corti-Organ in der Coiled Cochlea. Die Cristae und Maculae enthalten vestibulären Haarzellen, die mechanische Reize, um die besonderen Sinn für Ausgewogenheit dienstbar, während auditiven Haarzellen im Corti-Organ die primären Wandler für Hörgeräte sind ein transduzieren. Zellschicksals Spezifikation in diesen Sinnesepithelien und Morphogenese der Bogengänge und Hörschnecke finden während der zweiten Woche der Schwangerschaft in der Maus und sind weitgehend vor der Geburt 2,3 abgeschlossen. Entwicklungsstudien der Maus Innenohr werden routinemäßig von der Ernte transgenen Embryonen bei unterschiedlichen embryonalen oder postnatalen Stadien, um Einblick in die molekularen Grundlagen der zellulären und / oder morphologische Phänotypen gewinnen 4,5 durchgeführt. Wir vermuten, dass Gen-Transfer in die Entwicklungsländer Maus Innenohr in utero &lt;/ Em&gt; im Rahmen der Gewinn-und Verlust-of-function-Studien stellt einen kostenlosen Zugang zur traditionellen Maus-Transgenese für die Abfrage der genetischen Mechanismen, die Säugetier-Innenohr Entwicklung 6. Das experimentelle Paradigma zur Gen Fehlexpression Studien im sich entwickelnden Maus Innenohr führen demonstriert hier löst sich in drei Schritten: 1) ventrale Laparotomie; 2) transuterine Mikroinjektion, und 3) in vivo Elektroporation. Ventralen Laparotomie ist eine Maus Überleben chirurgische Technik, die Externalisierung der Gebärmutter ermöglicht, experimentelle Zugang zu den implantierten Embryonen 7 zu gewinnen. Transuterine Mikroinjektion ist die Verwendung von abgeschrägten, Glaskapillare Mikropipetten Expressionsplasmid in das Lumen des Ohr-Vesikel oder otocyst einzuführen. In-vivo-Elektroporation die Anwendung von Rechteckwellen, direkte Stromimpulse an Expressionsplasmid in Vorläuferzellen 8-10 anzutreiben. 11. Maus experimentellen embryologischen Techniken kann schwierig sein, von Prosa zu lernen und Standbilder allein. In der vorliegenden Arbeit zeigen wir, die 3 Schritte in der Gen-Transfer Verfahren. Die meisten kritisch, setzen wir digitale Video-Mikroskopie genau zeigen, wie man: 1) zu identifizieren Embryo Orientierung in utero, 2) neu zu orientieren Embryonen für die gezielte Injektionen an den otocyst; 3) microinject DNA mit Tracer-Farbstoff-Lösung in die otocyst gemischt an embryonalen Tag 11,5 und 12,5; 4) elektroporieren des eingespritzten otocyst und 5) Label elektroporiert Embryonen für postnatale Auswahl bei der Geburt. Wir bieten repräsentative Beispiele von erfolgreich transfizierten Innenohr; eine bildliche Anleitung zu den häufigsten Ursachen von otocyst Misstargeting; diskutieren, wie man gemeinsame methodische Fehler zu vermeiden; und Gegenwart Leitlinien für ein Schreiben in utero gEn-Transfer-Protokoll Tierpflege.

Watch this Scientific Journal Video about Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation at JoVE.com

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

Die Maus ist eine Innenohr-derived Plakode Sinnesorgan, dessen Entwicklungs-Programm wird während der Schwangerschaft erarbeitet. Wir definieren ein<em&gt; In der Gebärmutter</em&gt; Gentransfer-Technik, bestehend aus drei Schritten: Maus ventralen Laparotomie, transuterine Mikroinjektion, und<em&gt; In-vivo-</em&gt; Elektroporation. Wir nutzen digitale Video-Mikroskopie, um die kritischen experimentellen embryologischen Techniken zu demonstrieren.

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 ...

gene-transfer-to-developing-mouse-inner-ear-vivo

Research

JoVE Journal

Neuroscience

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

14.0K Aufrufe.  Oregon Health & Science University. Die Maus ist eine Innenohr-derived Plakode Sinnesorgan, dessen Entwicklungs-Programm wird während der Schwangerschaft erarbeitet. Wir definieren ein In der Gebärmutter Gentransfer-Technik, bestehend aus drei Schritten: Maus ventralen Laparotomie, transuterine Mikroinjektion, und In-vivo- Elektroporation. Wir nutzen digitale Video-Mikroskopie, um die kritischen experimentellen embryologischen Techniken zu demonstrieren.

Serie: Gene Transfer to the Developing Mouse Inner Ear by In Vivo 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 Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

In Utero intraventrikuläre Injektion und Elektroporation von E15 muriner Embryonen

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

Biologie

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Die Transfektion von Maus retinalen Ganglienzellen durch In vivo Elektroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Neurowissenschaften

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 ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

Maus in Utero Elektroporation: Kontrollierte Spatiotemporal Gene Transefection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and genetic mechanisms that contribute to hearing loss. Introduction of vectors into the inner ear must be done in a way that widely distributes the agent throughout the cochlea while minimizing injury to the existing structures. This manuscript describes a post-auricular surgical approach that can be used for mouse cochlear therapy using molecular, pharmacologic, and viral delivery to mice postnatal day 10 and older via the round window membrane (RWM). This surgical approach enables rapid and direct delivery into the scala tympani while minimizing blood loss and avoiding animal mortality. This technique involves negligible or no damage to essential structures of the inner and middle ear as well as neck muscles while wholly preserving hearing. To demonstrate the efficacy of this surgical technique, the vesicular glutamate transporter 3 knockout (VGLUT3 KO) mice will be used as an example of a mouse model of congenital deafness that recovers hearing after delivery of VGLUT3 to the inner ear using an adeno-associated virus (AAV-1).

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

Chirurgische Verfahren zum viral vermittelte Gentransfer in die Maus-Innenohr über die runde Fenstermembran

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...

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 ...

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.

A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

Direkte Lieferung von MIF Morpholinos Into the Zebrafisch Otocyst durch Injektion und Elektroporation Beeinflusst Inner Ear Entwicklung

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the ...

Gentransfer an sich entwickelnden Maus Innenohr

Zusammenfassung

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