Name: surgical method for virally mediated gene delivery to the mouse inner ear through the round window membrane
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This work is supported by an R21 grant from the National Institutes of Health and by a grant from Hearing Research, Incorporated.

MERK: Alle prosedyrer og dyr håndtering holdt NIH etiske retningslinjer og godkjent protokoll kravene i Institutional Animal Care og bruk komité ved University of California, San Francisco. 1. Klargjøre Animal for kirurgi <ol><li> Utføre kirurgiske prosedyrer i en ren, dedikert plass. Autoklav alle kirurgiske instrumenter, sterilisere med et glass-perle sterilisator før operasjonen. MERK: I denne protokollen, bruker postnatal dag 10-12 (P10-12) FVB mus. Ulike aldre og stammer a...

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I dette arbeidet, vi beskriver i detalj en teknikk som kan brukes for cochlea genterapi, med mål om å gjenopprette eller redde normal auditiv funksjon som er kompromittert av en genetisk defekt. Som det er typisk atraumatisk, er denne tilnærmingen trygg for cochlea genoverføring eller andre potensielle molekylære terapier 30. Andre tilnærminger for cochlea-terapi har blitt beskrevet, inkludert en ventral tilnærming 24, cochleostomy 31,32 og endolymfatiske sac levering 33</sup...

Genterapi har lenge vært foreslått som en potensiell behandling for genetisk hørselstap, men suksess på dette området har vært unnvikende en. Hittil har viralt medierte metoder dominerte grunn den teoretiske muligheten til å målrette bestemte celletyper innenfor den relativt utilgjengelig cochlea. Begge adenovirus (AV) og adenoassosiert virus (AAV) har vært brukt i cochlea genavlevering. AAVs er fordelaktige i sneglehuset for en rekke årsaker. De er replikering-mangel virus og effektivt kan overføre transgene molekyler til ulike celletyper, inkludert nevroner, et viktig mål for en rekke årsaker til hørselstap. AAV inntreden i cellen blir mediert av spesifikke reseptorer 2; Således må valget av en bestemt serotype være kompatibelt med de celletypene som skal omformes. AAVs effektivt kan transfektere hårceller 3 og innlemme i vertsgenomet, hvilket resulterer i stabile, langtids ekspresjon av transgenic protein og fenotypisk endring i cellen 4. Selv om ikke nødvendigvis en fordel for kortsiktige programmer som hår-celle gjenfødelse, er langsiktig uttrykk veldig viktig for stabil redning av genetiske defekter. Fordi AAVs ikke er forbundet med noen menneskelig sykdom eller infeksjon og demonstrere ingen ototoksisitet 5,6,7, de er en ideell kandidat for bruk i genterapi for nedarvede former for hørselstap 8. Overføring av eksogene genetisk materiale inn i pattedyr indre øret ved hjelp av virale vektorer er undersøkt de siste ti årene og fremstår som en lovende teknikk for behandling av både genetiske og ervervede former for hørselstap 9. Sneglehuset er potensielt et ideelt mål for genterapi av flere grunner: 1) sin lille volum nødvendiggjør en begrenset mengde av viruset nødvendig; 2) sin relative isolasjon fra andre organsystemer grenser bivirkninger; og 3) dets væskefylte kamre lette virallevering over hele labyrinten 10, 11,12,13,14, 15. Musemodeller av medfødt døvhet tillate bruk av mange metoder for studie for å overvåke utviklingen av det indre øret på en systematisk, replicable måte. Mens den lille størrelsen på muse cochleae ikke presentere noen kirurgisk vanskelighetsgrad, fungerer musen som en svært viktig modell for studiet av genetiske hørselstap, med flere eksperimentelle fordeler fremfor andre arter 16. Musemodeller tillate vurdering av en rekke egenskaper gjennom genetisk kobling analyse, innsamling av detaljerte morfologiske observasjoner, og simulere patogene scenarier; som sådan, de er gode kandidater for viralt mediert genterapi. Omfattende genetiske studier på mus kombinert med teknologiske fremskritt har gjort det mulig å generere genmodifiserte mus i en reproduserbar måte på tvers av laboratorier 17,18, 19, 20,21. Furthermore, det finnes mange modeller for både kjøpt og arvet hørselstap fenotyper hos mus, slik streng testing i denne dyremodell 22, 23,24. Dermed korrigere hørsel ved hjelp av viralt mediert genterapi i en musemodell er en passende første skritt i jakten på en kur for sykdom hos mennesker. Vi har tidligere vist at transgene mus som manglet vesicular glutamat Transporter 3 (VGLUT3) er født døv på grunn av mangel av glutamat utgivelse i IHC bånd synapse 25. Fordi denne mutasjonen ikke fører til en primær degenerasjon av den sensoriske hårceller, disse mutante mus er potensielt en utmerket modell for å teste cochlea genterapi for medfødt hørselstap. Hittil har en rekke virus leverings teknikker for cochlea genterapi blitt beskrevet, inkludert rundt vindu membran diffusjon, runde vinduet membran injeksjon, og levering via en cochleostomy. Det er potenteial fordeler og ulemper ved hver av disse tilnærmingene 9. Her rapporterer vi en kirurgisk metode for viralt genavlevering til VGLUT3 KO mus indre øret gjennom det runde vinduet membran (RWM). Den post-auricular RWM injeksjon metode er minimal invasiv med utmerket hørsel bevaring, og er relativt rask. Som vi tidligere har publisert, i et forsøk på å gjenopprette hørsel i denne musemodellen, ble en AAV1 vektor som bærer genet VGLUT3 (AAV1-VGLUT3) føres inn i sneglehuset av disse døve mus ved postnatal dag 12 (P! @), Noe som resulterer i restaurering av å høre 26. Hørsel i VGLUT3 KO mus ble verifisert av auditive hjernestammen respons (ABR), mens transgene protein uttrykk ble bekreftet ved hjelp immunfluorescens (IF). Denne metodikken demonstrerer dermed at viralt-mediert genterapi kan korrigere en genetisk feil som ellers ville resulterer i døvhet.

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			<td style="width:177px;">World precision instruments, inc.</td>
			<td style="width:161px;">1B100F-4</td>
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			<td height="24" style="height:24px;width:355px;">Tissue glue (Vetcode)</td>
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			<td height="24" style="height:24px;width:355px;">Rabbit Anti-GFP antibody</td>
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			<td height="20" style="height:20px;">Dissecting microscope&#160;&#160;&#160;&#160;&#160;</td>
			<td>Leica</td>
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			<td>MZ95</td>
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			<td height="20" style="height:20px;">Flaming/ Brown Micropipette&#160;&#160;&#160;&#160;&#160;</td>
			<td>Sutter Instrument Co</td>
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			<td height="20" style="height:20px;">Puller Model P-97&#160;&#160;</td>
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			<td height="20" style="height:20px;">TDT BioSig III System&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>&#160;Tucker-Davis Technologies</td>
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surgical method for virally mediated gene delivery to the mouse inner ear through the round window membrane

Genterapi, som brukes for å oppnå funksjonell utvinning fra sensorineurale døvhet, lover å gi bedre forståelse av de underliggende molekylære og genetiske mekanismer som bidrar til hørselstap. Innføring av vektorer inn i det indre øret må gjøres på en måte som er mye distribuerer midlet gjennom hele sneglehuset samtidig minimere skade på det eksisterende strukturer. Dette manuskriptet beskriver en post-auricular kirurgiske tilnærming som kan brukes for mus cochlea terapi bruker molekylære, farmakologisk, og viral levering til mus postnatal dag 10 og eldre via det runde vinduet membran (RWM). Det kirurgiske tilnærming muliggjør rask og direkte levering i scala tympani samtidig minimere blodtap og unngå dødelighet dyr. Denne teknikken innebærer ubetydelig eller ingen skade på viktige strukturer i indre og mellomøret samt nakkemusklene mens helt bevare hørselen. For å demonstrere effekten av dette kirurgisk teknikk, vesicular glutamspiste transporter 3-knockout (VGLUT3 KO) mus blir brukt som et eksempel på en musemodell av medfødt døvhet som gjenoppretter høre etter levering av VGLUT3 til det indre øret ved hjelp av en adenoassosiert virus (AAV-1).

Å kontrollere de tekniske funksjonene og nytten av post-aurikulær tilnærming for cochlea molekylær terapi, ble AAV1-VGLUT3, AAV1-GFP og AAV2-GFP levert inn P10-12 mus indre øret via RWM. Denne tilnærmingen viser vellykket transgene uttrykk innenfor indre hårcellene (IHC) (VGLUT3 Figur 1 og GFP Figur 2 og GFP figur 3A), ytre hårcellene (OHC) (GFP figur 2) og støtteceller (GFP figur 2 og figur 3A) 26 u...

Watch this Scientific Journal Video about Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane at JoVE.com

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

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.

Kirurgisk metode for viralt genavlevering til Mouse Inner Ear gjennom det runde vinduet Membrane

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

Research

JoVE Journal

Neuroscience

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

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

Isolere LacZ-uttrykke Celler fra Mouse Inner Ear Vev bruker flowcytometrisystemer

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 ...

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

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

Genoverføring til Developing Mouse indre øret ved<em&gt; In Vivo</em&gt; 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 ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

En polert og forsterket tynnet-skalle Window for langsiktig Imaging av Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch Clamp Recordings i Indre ørehår celler isolert fra sebrafisk

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  ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

Utnytte Kombinert Metoder for å definere rollen til plasmamembranen Levering Under Axon Forgrening og neuronal morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Mus Microsurgery Infusion Teknikk for målrettet Stoff Levering inn i CNS<i&gt; via</i&gt; Arteria carotis interna

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

Laminectomy og vindus kabel i mus

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...