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

Dette arbejde blev støttet af afdelingen for Egne Forskning ved National Institute on Døvhed og andre kommunikationsteknologier Disorders. Yderligere støtte blev leveret af NIDCD 5R01 DC007613.

1. Utricle Dissektion og Kultur <ol><li> Afliv en voksen (4 uger gamle eller ældre) mus ved hjælp af en godkendt protokol og halshugge. </li><li> Røverkøb den ydre øregang på begge sider af hovedet og trække huden frem mod næsen. Gennemskære hovedet fra tilbage til forsiden og fjerne hjernen fra begge sider for at afsløre knoklet labyrint. </li><li> Trim kraniet væk fra benet cochlea og overføre den benede labyrint (herunder bulla) til en vævskultur hætte forsynet med et dissektionsmikroskop (Bemærk: ad...

<ol>
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Sansehårceller er modtagelige for død forårsaget af en række spændinger, herunder ældning, støj trauma, og udsættelse for ototoksiske lægemidler, herunder aminoglykosid antibiotika og det antineoplastiske middel cisplatin. Hos pattedyr hår celledød resulterer i permanent høretab og / eller balance forstyrrelser. In vitro modelsystemer er kritiske værktøjer til undersøgelser med henblik på fastlæggelse af de cellulære og molekylære mekanismer bag hår celledød, såvel som dem, der sigter på ...

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; Firma</strong</td&gt;<td<strong&gt; Katalog nr.</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 tænger (# 55)</td&gt;<td&gt; Gode Science Tools</td&gt;<td&gt; 11255-20</td&gt;</tr&gt;<tr&gt;<td&gt; Fin pincet (# 5)</td&gt;<td&gt; Gode Science Tools</td&gt;<td&gt; 11252-30</td&gt;</tr&gt;<tr&gt;<td&gt; Tang (# 3)</td&gt;<td&gt; Gode Science Tools</td&gt;<td&gt; 11231-30</td&gt;</tr&gt;<tr&gt;<td&gt; Penicillin G</td&gt;<td&gt; Sigma</td&gt;<td&gt; P3032</td&gt;</tr&gt;<tr&gt;<td&gt; Føtalt bovint serum</td&gt;<td&gt; Invitrogen</td&gt;<td&gt; 10082-139</td&gt;</tr&gt;<tr&gt;<td&gt; Nunc mini-bakken</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 afkalker</td&gt;<td&gt; Fisher</td&gt;<td&gt; CS510-1D</td&gt;</tr&gt;<tr&gt;<td&gt; Natriumborhydrid</td&gt;<td&gt; Sigma</td&gt;<td&gt; 452882</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-Myosin 7a (polyklonale)</td&gt;<td&gt; Proteus Biosciences</td&gt;<td&gt; 25-6790</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-Myosin 7a (monoklonalt)</td&gt;<td&gt; Developmental Studies Hybridoma Bank</td&gt;<td&gt; MYO7A 138-1</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-Calmodulin</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;. Reagenser og værktøjer, der anvendes i utricle kultur og adenovirusinfektion.</p&gt;<p class="jove_content"&gt; * Bemærk: Ad-GFP er normalt ved Vector Biolabs på et lager titer på 1x10<sup&gt; 10</sup&gt; PFU / ml. Vi ønskede et brugerdefineret amplifikation med henblik på at tilvejebringe stamvirus ved en titer på 1,2 x 10<sup&gt; 11</sup&gt; PFU / ml.</p&gt;

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

Høretab og balance forstyrrelser er ofte forårsaget af død mechanosensory hårceller, som er de receptor celler i det indre øre. Da der ikke er cellelinje, der på tilfredsstillende betegner mammale hårceller, forskning hårceller afhængig primære organkulturer. Den bedst karakteriseres in vitro model system af modent pattedyrprotein hårceller udnytter organkulturer af utricles fra voksne mus (figur 1) 1-6. Det utricle er en vestibulære organ, og hårceller i utricle er ens i både struktur og funktion til at hårcellerne i det auditive organ, orglet af Corti. Den voksne mus utricle præparatet repræsenterer en moden sensorisk epithel til undersøgelser af den molekylære signaler, som regulerer overlevelsen, homeostase, og disse cellers død. Mammale snegle-hårceller er terminalt differentierede og ikke regenereres, når de går tabt. I ikke-mammaliske hvirveldyr, auditive eller vestibulære hår celledød efterfølges af en robust regenerering, der genskaber hørelse og balance funktioner 7, 8. Hårcelleregeneration medieres af glia-lignende understøtter celler, som kontakter de basolaterale overflade af hårceller i det sensoriske epithel 9, 10. Støtte-celler er også vigtige mediatorer af hår celleoverlevelse og død 11. Vi har for nylig udviklet en teknik til infektion af støtte celler i dyrkede utricles med adenovirus. Ved hjælp af adenovirus type 5 (dE1/E3) at afgive et transgen, der indeholder GFP under kontrol af CMV-promotoren, finder vi, at adenovirus specifikt og effektivt inficerer støtte-celler. Støtte celleinfektion effektivitet er ca 25-50%, og hårceller ikke inficeret (figur 2). Vigtigt, vi finder, at adenovirusinfektion understøtte celler ikke resulterer i toksicitet til hårceller eller støtter celler, som celletal i Ad-GFP infected utricles svarer til dem i ikke-inficerede utricles (Figur 3). Således adenovirus-medieret genekspression i støtte celler fra dyrkede utricles giver et kraftfuldt værktøj til at studere de roller støtte celler som mediatorer af hår celle overlevelse, død og genfødsel.

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

Mechanosensory hårceller er receptor celler i det indre øre. Den bedst karakteriseres<em&gt; In vitro</em&gt; Modelsystem af modne pattedyr hårceller udnytter organkulturer af utricles fra voksne mus. Vi præsenterer dissektion af voksne mus utricle, og vi viser adenovirus-medieret infektion af støtte-celler i dyrkede utricles.

Dissektion af voksne mus Utricle og adenovirus-medieret støtte-celleinfektion

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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