Name: dissection of adult mouse utricle and adenovirus-mediated supporting-cell infection
Uploaded: 2012-03-28T12:00:00
Description: Watch this Scientific Journal Video about Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection at JoVE.com

Dit werk werd ondersteund door de afdeling van de intramurale onderzoek aan het National Institute on Doofheid en Andere Communicatie Wanorde. Extra ondersteuning werd verleend door het NIDCD, toont 5R01 DC007613.

1. Utricle Dissection en Cultuur <ol><li> Euthanaseren van een volwassene (4 weken oud of ouder) muis met behulp van een goedgekeurd protocol en te onthoofden. </li><li> Knip de uitwendige gehoorgang aan beide zijden van het hoofd en trek de huid naar de neus. Halveren het hoofd van achter naar voren en verwijder de hersenen van beide zijden om de benige labyrint te onthullen. </li><li> Knip de schedel uit de buurt van het benige slakkenhuis en de benige labyrint (inclusief de bulla) om een ​​weefselkweek kap uitg...

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Haarcellen zijn gevoelig voor de dood veroorzaakt door een veelheid van spanningen, zoals vergrijzing, lawaai trauma, en blootstelling aan ototoxische geneesmiddelen, met inbegrip van het aminoglycoside-antibiotica en het antineoplastische middel cisplatine. Bij zoogdieren haar celdood leidt tot blijvend gehoorverlies en / of balans verstoring. In vitro model systemen zijn essentiële hulpmiddelen voor studies die gericht zijn op het bepalen van de cellulaire en moleculaire mechanismen die ten grondslag liggen ...

A correction was made to <a href="http://www.jove.com/video/3734">Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection</a>. There was an error with a figure, the authors middle names were omitted, and the acknowledgements were updated.

Erratum: Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; Reagens</strong</td&gt;<td<strong&gt; Bedrijf</strong</td&gt;<td<strong&gt; Catalogus nummer</strong</td&gt;</tr&gt;<tr&gt;<td&gt; Medium 199</td&gt;<td&gt; Gibco / Invitrogen</td&gt;<td&gt; 12350</td&gt;</tr&gt;<tr&gt;<td&gt; DMEM/F12</td&gt;<td&gt; Gibco / Invitrogen</td&gt;<td&gt; 11320</td&gt;</tr&gt;<tr&gt;<td&gt; Fine pincet (# 55)</td&gt;<td&gt; Fine Science Tools</td&gt;<td&gt; 11255-20</td&gt;</tr&gt;<tr&gt;<td&gt; Fine pincet (# 5)</td&gt;<td&gt; Fine Science Tools</td&gt;<td&gt; 11252-30</td&gt;</tr&gt;<tr&gt;<td&gt; Tang (# 3)</td&gt;<td&gt; Fine Science Tools</td&gt;<td&gt; 11231-30</td&gt;</tr&gt;<tr&gt;<td&gt; Penicilline G</td&gt;<td&gt; Sigma</td&gt;<td&gt; P3032</td&gt;</tr&gt;<tr&gt;<td&gt; Foetaal runderserum</td&gt;<td&gt; Invitrogen</td&gt;<td&gt; 10082-139</td&gt;</tr&gt;<tr&gt;<td&gt; Nunc mini-tray</td&gt;<td&gt; Fisher</td&gt;<td&gt; 12-565-68</td&gt;</tr&gt;<tr&gt;<td&gt; Adenovirus-GFP</td&gt;<td&gt; Vector Biolabs</td&gt;<td&gt; 1060 *</td&gt;</tr&gt;<tr&gt;<td&gt; Cal-Ex ontkalker</td&gt;<td&gt; Fisher</td&gt;<td&gt; CS510-1D</td&gt;</tr&gt;<tr&gt;<td&gt; Natriumboorhydride</td&gt;<td&gt; Sigma</td&gt;<td&gt; 452.882</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-Myosine 7a (polyklonaal)</td&gt;<td&gt; Proteus Biosciences</td&gt;<td&gt; 25-6790</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-Myosine 7a (monoklonale)</td&gt;<td&gt; Developmental Studies Hybridoma Bank</td&gt;<td&gt; MYO7A 138-1</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-Calmoduline</td&gt;<td&gt; Sigma</td&gt;<td&gt; C 3545</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-Calbindin</td&gt;<td&gt; Chemicon</td&gt;<td&gt; AB1778</td&gt;</tr&gt;<tr&gt;<td&gt; Fluoromount G</td&gt;<td&gt; Southern Biotechnology Associates</td&gt;<td&gt; 0100-01</td&gt;</tr&gt;</tbody&gt;</table&gt;<p class="jove_content"<strong&gt; Tabel 1</strong&gt;. Reagentia en instrumenten die in utricle cultuur en adenovirus infectie.</p&gt;<p class="jove_content"&gt; * Opmerking: Ad-GFP wordt normaliter door Vector Biolabs op een voorraad titer van 1x10<sup&gt; 10</sup&gt; Pfu / ml. Wij gevraagd een aangepaste amplificatie om de voorraad virus voorzien op titer 1,2 x10<sup&gt; 11</sup&gt; Pfu / ml.</p&gt;

dissection of adult mouse utricle and adenovirus-mediated supporting-cell infection

Gehoorverlies en evenwichtsstoornissen worden vaak veroorzaakt door de dood van mechanosensorische haarcellen, die de receptor cellen van het binnenoor. Omdat er geen cellijn die naar tevredenheid is zoogdier haarcellen, onderzoek naar haarcellen is gebaseerd op primaire orgaan culturen. De beste kenmerk vitro modelsysteem van volwassen zoogdieren haarcellen gebruikt orgaankweken van utricles van volwassen muizen (figuur 1) 1-6. De utricle is een vestibulaire orgaan, en de haarcellen van het utricle zijn vergelijkbaar in zowel structuur als functie om de haarcellen in het gehoororgaan, het orgaan van Corti. De volwassen muis utricle voorbereiding is een volwassen sensorische epitheel voor de studie van de moleculaire signalen die het overleven, homeostase, en de dood van deze cellen te reguleren. Zoogdieren cochleaire haarcellen zijn terminaal gedifferentieerd en niet wordt geregenereerd wanneer ze verloren zijn. In niet-mamMalinese gewervelde dieren, auditieve of vestibulaire haar celdood wordt gevolgd door robuuste regeneratie die gehoor en evenwicht functies 7, 8 herstelt. Hair celregeneratie wordt gemedieerd door glia-achtige ondersteunende cellen, die de basolaterale oppervlak van haarcellen contact op te nemen in de sensorische epitheel 9, 10. Ondersteunende cellen zijn ook belangrijke mediatoren van haar overleving van de cel en de dood 11. We hebben onlangs een methode voor infectie van cellen in cultuur ondersteunen utricles met adenovirus. Met adenovirus type 5 (dE1/E3) een transgen met GFP onder de controle van de CMV promoter leveren wij dat adenovirus specifiek en efficiënt infecteert ondersteunende cellen. Steun cel infectie-efficiëntie ongeveer 25-50% en haarcellen niet geïnfecteerd (figuur 2). Belangrijk vinden we dat adenovirale infectie van ondersteunende cellen niet leidt tot toxiciteit voor haarcellen of ondersteunende cellen, zoals cellen in Ad-GFP infected utricles gelijkwaardig zijn aan die in niet-geïnfecteerde utricles (figuur 3). Zo adenovirus-gemedieerde genexpressie in de ondersteuning van cellen van gekweekte utricles biedt een krachtig instrument om de rol van ondersteunende cellen als mediatoren van haar overleving van de cel, de dood en regeneratie te bestuderen.

Watch this Scientific Journal Video about Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection at JoVE.com

Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection

Mechanosensorische haarcellen zijn de receptor cellen van het binnenoor. De meest kenmerk<em&gt; In vitro</em&gt; Modelsysteem van volwassen zoogdier haarcellen maakt gebruik van orgaan culturen van utricles van volwassen muizen. We presenteren de dissectie van de volwassen muis utricle, en tonen we aan adenovirus-gemedieerde infectie van ondersteunende cellen in gekweekte utricles.

Dissectie van volwassen muis Utricle en Adenovirus-gemedieerde Ondersteunende-cel infectie

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear.  Since there ...

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