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

13.9K 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

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Gene Lieferung in Postnatale Rattengehirn von Non-ventrikuläre Plasmid Injektion und Elektroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Forschung

Neurowissenschaften

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 ...

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

Die Quantifizierung der Aktivität von Cis-Regulatorische Elemente in der Maus Retina durch Explantation Elektroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

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 ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

Isolating LacZ-exprimierenden Zellen aus Maus Innenohrgeweben mittels Durchflusszytometrie

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

Hochauflösende Live-Bildgebung von Zell-Verhalten in der Entwicklung von Neuroepithel

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

Patch-Clamp-Aufnahmen im inneren Haarsinneszellen Isoliert von Zebrafisch

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

Genetische Manipulation der Maus entwickeln Hypothalamus durch<em&gt; In utero</em&gt; Elektroporation

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

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 ...