We would like to acknowledge Dr. Bradley Schulte for comments on this protocol. This work was supported by National Institutes of Health grant R00 (5R00DC010220). This project was performed in a renovated laboratory space supported by Grant C06RR014516.

NOTA: Tutti i protocolli che impiegano animali vivi deve essere esaminato e approvato da una cura e l&#39;uso degli animali Comitato Istituzionale (IACUC) e deve seguire i metodi per la cura e l&#39;uso di animali da laboratorio ufficialmente approvato. Tutte le dissezioni devono essere eseguiti con tecnica sterile su un banco a flusso laminare pulita. Guanti e una maschera, se desiderato, devono essere indossati durante questa procedura. 1. La dissezione del Ear embrionali di topo interno <ol><li> Impostazione per organo di culture espianto Corti: <ol><li> Sterilizzare la cappa coltura tissutale flusso laminare accendendo la luce UV per circa 30 minuti e disinfettare la superficie con etanolo al 70% prima dell&#39;uso. Inoltre, sterilizzare tutti gli strumenti di dissezione tra cui strumenti di dissezione raffinati e piatti Sylgard rivestite di embrione dissezione tramite autoclave o per immersione in etanolo al 70% per almeno 20 minuti prima dell&#39;uso. Eliminare il 70% di etanolo e lasciare i piatti e gli strumenti per aria secca in un flusso laminare Clea puliton panchina prima dell&#39;uso. </li><li> Preparare Balanced Salt Solution sterile di Hank (HBSS) / (4- (2-idrossietil) -1-acido piperazineethanesulfonic) (HEPES) miscela aggiungendo 10% di 10x HBSS e 0,5% HEPES e regolare il pH a circa 7,2 e sterilizzare il filtro soluzione finale e conservare a 4 ° C. Eseguire tutte le dissezioni di refrigerate soluzione HBSS / HEPES per conservare meglio il tessuto durante tutta la procedura. </li><li> Piatti fondo di vetro a matrice Coat membrana basale aggiungendo 5 ml di freddo (4 ° C), sterile di Dulbecco modificato (DMEM) per un&#39;aliquota di 300 ml di matrice membrana basale in una sterile 15 ml tubo di polipropilene. Mescolare vortexando contenuti e aggiungere 150 microlitri della miscela di matrice DMEM membrana basale al centro di ciascuna piastra di coltura in modo da coprire l&#39;intera cultura ben presente al centro del piatto. Coprire i piatti e conservare in un 37 ° C incubatore per almeno 45 minuti prima dell&#39;uso. </li><li> Versare 4-5 ml refrigerate HBSS in three sterili piatti polistirene Petri e lasciare chiusi nella cappa. </li><li> Preparare mezzo di coltura in una provetta da 15 ml di polipropilene sterile aggiungendo 9 ml di DMEM, 100 ml di integratori N2, 10 g / ml Cipro e 1 ml di FBS. </li></ol></li><li> Isolamento di embrioni: NOTA: In questa procedura per la coltura di espianti cocleari da topi embrionali, raccolta orecchie interne dal giorno embrionale (E) 13 ossa temporali con il giorno dopo la fecondazione essere considerati come E1 9. Il protocollo può essere utilizzato anche con il mouse embrionale orecchio interno a partire dal E12 a E18. <ol><li> Euthanize un mouse a tempo-incinta di CO 2 e sacrificare animali per dislocazione cervicale o appropriati protocolli approvati. Eseguire euthanization dalla cappa coltura tissutale a flusso laminare per mantenere la sterilità in sala coltura di tessuti. </li><li> Posizionare il mouse eutanasia sul tovagliolo di carta con l&#39;addome rivolto verso l&#39;alto e disinfettare l&#39;addome con il 70% di etanolo. </li><li> Aprire la cavità addominale afferrando la pellecon pinze curve con una mano e dell&#39;epidermide recisi e muscolo lungo la linea mediana con le forbici con un&#39;altra mano. Fare due tagli perpendicolari su entrambi i lati e tirare fuori l&#39;utero attentamente sollevando corna uterine bilaterali con una pinza e staccarla da sotto le forbici del tessuto connettivo con. </li><li> Posizionare la catena embrionale in una capsula di Petri contenente soluzione raffreddata dissezione (miscela HBSS / HEPES) e trasferimento al banco pulito a flusso laminare. </li><li> Rimuovere gli embrioni con attenzione di placenta e metterli in una delle piastre sterili contenenti soluzione dissezione. Se la soluzione diventa sanguinosa, spostarli in una nuova piastra di Petri. NOTA: A questo punto, gli embrioni possono essere messo in scena con una guida messa in scena Theiler. </li><li> Decapitare embrioni pizzicando alla regione del collo con un paio di pinze o forbici pulite e posizionare le teste in un piatto fresco Petri contenente soluzione dissezione raffreddata. </li></ol></li><li> La dissezione delle orecchie interne: <ol><li> Luogo one testa embrione in un piatto Sylgard sterile contenente dissezione a freddo </li><li> soluzione. Lavorando sotto un microscopio da dissezione con un ingrandimento di 1,6X ~, immobilizzare la testa dell&#39;embrione posizionando perni Minutien intorno o più vicino alla zona degli occhi (Figura 1A). </li><li> Utilizzando due coppie di pinze sterili rimuovere con attenzione la pelle e aprire il cranio sul lato dorsale lungo la linea mediana (Figura 1B). Rimuovere cervello dalla cavità cranica e orecchie interne situate all&#39;interno ossa temporali possono essere identificate in questa fase (Figura 1C, D). Il rivestimento del vaso sanguigno, che di solito è presente intorno l&#39;orecchio interno, può essere utilizzato come punto di riferimento per identificare l&#39;orecchio interno in questa fase (Figura 1D). </li><li> Sezionare l&#39;orecchio interno dalle ossa temporali posizionando la pinza sotto il tessuto e isolando l&#39;orecchio interno dalla base del cranio e trasferimento in un piatto contenente soluzione di dissezione a freddo (Figura 1E, F). </li></ol></li></ol> 2. Generazione di Organo delle Culture Corti Explant NOTA: In questa fase di sviluppo, il tessuto è cartilagineo e può essere facilmente sezionato con pinze. <ol><li> Orient l&#39;orecchio interno in modo che parte ventrale è rivolto verso l&#39;alto (Figura 2A) e stabilizzare l&#39;orecchio interno inserendo dolcemente la punta di perni Minutien attraverso la porzione vestibolare dell&#39;orecchio interno (Figura 2B). </li><li> Uso pinze ultrafini tagliare aprire la cartilagine sovrastante facendo un&#39;incisione con una estremità di pinze vicino alla finestra ovale e rimuovere la cartilagine dalla coclea (Figura 2C) con attenzione. NOTA: È importante assicurarsi che pinze non sono inserite troppo profondamente nella cartilagine cartilagine sovrastante è talvolta fuso con il condotto cocleare nel qual caso è utile usare pinza chiusa per rilasciare la cartilagine dal condotto cocleare sottostante raschiando sotto la surface della cartilagine. A questo stadio di sviluppo, la spirale cocleare è solo tre quarti di giro in lunghezza. </li><li> Successivamente, esporre dell&#39;epitelio sensoriale posizionando la pinza alla regione preferita del condotto cocleare, sia la base o per lo all&#39;apice, e tirando delicatamente il tetto della coclea (Figura 2D). NOTA: Il tetto della coclea deve essere completamente rimosso in quanto può mascherare dell&#39;epitelio sensoriale ostacolare l&#39;analisi. NOTA: Questa procedura deve essere eseguita delicatamente come è facile da strappare dell&#39;epitelio sensoriale. </li><li> Come ultima fase, rimuovere con attenzione il tessuto connettivo sottostante dall&#39;epitelio sensoriale esposto modo che la base del trapianto cocleare è uniformemente piana. </li><li> Isolare la coclea sezionato dalla parte vestibolare dell&#39;orecchio interno con pinze. Questo può essere eseguito prima o dopo la fase 2.4 (figura 2E). </li><li> Trasferire l&#39;epitelio sensoriale cocleare sezionato in un seminterrato membrane matrix-rivestita cultura e con una paletta sterile 1,5 millimetri (Figura 2F). </li><li> Orient espianto cocleare con la superficie luminale dell&#39;epitelio rivolto verso l&#39;alto e aspirare la membrana basale soluzione di matrice-DMEM per appiattire accuratamente il tessuto e sostituirlo con 150 ml di terreno di coltura fresco aggiungendo delicatamente al piatto. Si deve prestare attenzione per assicurarsi che ogni espianto aderisce bene alla coprioggetto vetro rivestito e non sta galleggiando nel mezzo di coltura. Assicurarsi che la punta della pinza è rivolta verso l&#39;alto, mentre apponendo sul bene di vetro per evitare di danneggiare l&#39;espianto cocleare. </li><li> Trasferire delicatamente il piatto di cultura in un 150 millimetri piatto di coltura sterile e posto in un tessuto culturale incubatore a 37 ° C con il 5% di CO 2 per il periodo di tempo desiderato, di solito 3-6 giorni in vitro (DIV). Dopo 1 DIV, esaminare le culture sotto un microscopio da dissezione per assicurarsi che l&#39;espianto cocleare aderisce bene al piatto di coltura. </li><li> Dopo incubation in vitro per periodi di tempo desiderato, le culture sono trattati per immunoistochimica. NOTA: Le culture espianto di solito vengono incubate per 6 DIV che è equivalente a stadio di sviluppo, P0. Dopo incubazione in vitro, le culture possono essere trattati per altre applicazioni a valle come immunoistochimica (Figura 3B), RT-PCR, ibridazione in situ, e / o Western Blot. </li></ol> 3. Elettroporazione mediata Gene Transfer <ol><li> Configurazione per elettroporazione: <ol><li> Sterilizzare uno 100 millimetri vetro piatto Sylgard rivestite in autoclave o immersione in etanolo al 70% per circa 20 minuti e lasciare asciugare in un banco pulito prima dell&#39;uso. </li><li> Preparare DNA da un vettore di espressione di scelta utilizzando opportuni kit simo o midi-preparazione. I vettori di espressione utilizzati in questo protocollo sono pIRES2-Atoh1.EGFP e pCLIG-NeuroD1.EGFP. La concentrazione finale di DNA deve essere di almeno 1 mg / mL in DNA sterile /Senza RNAse acqua. </li></ol></li><li> Electroporating DNA in espianti cocleari embrionali: <ol><li> Aggiungere 10 ml di soluzione di DNA plasmidico da 100 mm a piatto Sylgard rivestite fresco e trasferire un espianto cocleare completamente sezionato (ottenuto dalla fase 2.5) nella soluzione di DNA. </li><li> Con superficie luminale dell&#39;epitelio rivolto verso l&#39;alto, inclinare la coclea leggermente in modo che sia perpendicolare al piano del piatto. </li><li> Collocare le piastre di elettrodi su entrambi i lati della coclea con paddle negativo (catodo) verso l&#39;epitelio sensoriale e la pala positivo (anodo) adiacente alla base della coclea. NOTA: Poiché DNA è caricato negativamente, migra da negativo a elettrodo positivo e inserendo l&#39;estremità del catodo della sonda adiacente dell&#39;epitelio sensoriale; il DNA principalmente passare attraverso la superficie dell&#39;epitelio sensoriale. </li><li> Utilizzando elettroporatore, consegnare 9 a 10 impulsi di 24 mV, 30 msec durata dell&#39;impulso con l&#39;interruttore a pedale su tegli elettroporatore e aggiungere 100 ml di terreno di coltura caldo alla coclea elettroporate. </li><li> Ripetere i passaggi 3.2.1-4 per tutti gli espianti cocleari e per il DNA di tutti i geni di interesse e di trasferire il coclee elettroporate nel piatto di matrice rivestite membrana basale per la placcatura (passo 2,7). Culture tutte le coclee elettroporate a 37 ° C, 5% CO 2 incubatore umidificato per lunghezze desiderate di tempo che sarà poi elaborati per immunocitochimica. NOTA: La coclea da un&#39;intera cucciolata di CD1 mouse (circa 8-12 cuccioli) può essere isolato, microdissezione, elettroporate e placcato in &lt;4 ore. </li></ol></li></ol> 4. Analisi delle Culture Cochlear Explant NOTA: Le colture vengono incubate per 6 solito DIV che è equivalente a stadio di sviluppo, P0. Dopo incubazione in vitro, le culture sono fissi e trattati per immunocitochimica. <ol><li> Dopo lunghezza desiderata di incubation, rimuovere terreno di coltura e rapidamente risciacquare l&#39;espianto cocleare in soluzione salina tamponata con fosfato (PBS) e incubare in 4% paraformaldeide (preparato in PBS) per 15 minuti a temperatura ambiente. </li><li> Lavare il fissativo aggiungendo PBS e sciacquare in PBS per tre volte per 15 min. </li><li> Permeabilize con PBS-T (PBS + 0,5% Tween) per 30 minuti prima di bloccare con PBS-T contenente siero di capra 10%. </li><li> Incubare in soluzione anticorpo primario contenente anticorpo primario in PBS-T con siero di capra 1% notte a 4 ° C. </li><li> Lavare la coclea tre volte a temperatura ambiente utilizzando PBS-T 15 min ciascuna prima di aggiungere anticorpi secondari coniugati Alexa appropriate. </li><li> Dal momento che l&#39;espianto cocleare è colta sul vetrino di vetro può essere gestito individualmente isolando il vetrino dal piatto e montaggio su una diapositiva. Ciò può essere ottenuto immergendo il piatto di coltura in OS30 solvente per almeno 1 ora a temperatura ambiente. Utilizzando la lama di rasoio sterile, staccare il vetrino dal dish e montare su un vetrino e visualizzare sotto un microscopio a fluorescenza. </li></ol>

<ol>
	<li>Sobkowicz, H. M., Bereman, B., Rose, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+development+of+the+organ+of+Corti+in+culture.">Organotypic development of the organ of Corti in culture.</a> J Neurocytol. 4 (5), 543-572 (1975).</li><li>Zheng, J. L., Gao, W. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+Math1+induces+robust+production+of+extra+hair+cells+in+postnatal+rat+inner+ears.">Overexpression of Math1 induces robust production of extra hair cells in postnatal rat inner ears.</a> Nat Neurosci. 3 (6), 580-586 (2000).</li><li>Zheng, J. L., Shou, J., Guillemot, F., Kageyama, R., Gao, W. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hes1+is+a+negative+regulator+of+inner+ear+hair+cell+differentiation.">Hes1 is a negative regulator of inner ear hair cell differentiation.</a> Development. 127 (21), 4551-4560 (2000).</li><li>Woods, C., Montcouquiol, M., Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Math1+regulates+development+of+the+sensory+epithelium+in+the+mammalian+cochlea.">Math1 regulates development of the sensory epithelium in the mammalian cochlea.</a> Nat Neurosci. 7 (12), 1310-1318 (2004).</li><li>Jones, J. M., Montcouquiol, M., Dabdoub, A., Woods, C., Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+differentiation+and+DNA+binding+(Ids)+regulate+Math1+and+hair+cell+formation+during+the+development+of+the+organ+of+Corti.">Inhibitors of differentiation and DNA binding (Ids) regulate Math1 and hair cell formation during the development of the organ of Corti.</a> J Neurosci. 26 (2), 550-558 (2006).</li><li>Dabdoub, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox2+signaling+in+prosensory+domain+specification+and+subsequent+hair+cell+differentiation+in+the+developing+cochlea.">Sox2 signaling in prosensory domain specification and subsequent hair cell differentiation in the developing cochlea.</a> Proc. Natl. Acad. Sci. U.S.A. 105 (47), 18396-18401 (2008).</li><li>Doetzlhofer, A., Basch, M. L., Ohyama, T., Gessler, M., Groves, A. K., Segil, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hey2+regulation+by+FGF+provides+a+Notch-independent+mechanism+of+maintaining+pillar+cell+fate+in+the+organ+of+Corti.">Hey2 regulation by FGF provides a Notch-independent mechanism of maintaining pillar cell fate in the organ of Corti.</a> Dev Cell. 16 (1), 58-69 (2009).</li><li>Puligilla, C., Dabdoub, A., Brenowitz, S. D., Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox2+induces+neuronal+formation+in+the+developing+cochlea.">Sox2 induces neuronal formation in the developing cochlea.</a> J Neurosci. 20 (2), 714-722 (2010).</li><li>Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Atlas of Mouse Development. , (1995).</li><li>Sher, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+embryonic+and+postnatal+development+of+the+inner+ear+of+the+mouse.">The embryonic and postnatal development of the inner ear of the mouse.</a> Acta Otolaryngol Suppl. 285, 1-77 (1971).</li><li>Torres, M., Giraldez, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+the+vertebrate+inner+ear.">The development of the vertebrate inner ear.</a> Mech Dev. 71 (1-2), 5-21 (1998).</li><li>Brown, S. T., Martin, K., Groves, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+inner+ear+induction.">Molecular basis of inner ear induction.</a> Curr Top Dev Biol. 57, 115-119 (2003).</li><li>Puligilla, C., Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+the+world&#8217;s+best+hearing+aid;+regulation+of+cell+fate+in+the+cochlea.">Building the world&#8217;s best hearing aid; regulation of cell fate in the cochlea.</a> Curr Opin Genet Dev. 19 (4), 368-373 (2009).</li><li>Wu, D. K., Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+inner+ear+development.">Molecular mechanisms of inner ear development.</a> Cold Spring Harb Perspect Biol. 4 (8), a008409 (2012).</li><li>Holt, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral-mediated+gene+transfer+to+study+the+molecular+physiology+of+the+mammalian+inner+ear.">Viral-mediated gene transfer to study the molecular physiology of the mammalian inner ear.</a> Audiol Neurootol. 7 (3), 157-160 (2002).</li><li>Stone, I. M., Lurie, D. I., Kelley, M. W., Poulsen, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus-mediated+gene+transfer+to+hair+cells+and+support+cells+of+the+murine+cochlea.">Adeno-associated virus-mediated gene transfer to hair cells and support cells of the murine cochlea.</a> Mol Ther. 11 (6), 843-848 (2005).</li></ol>

No conflicts of interest declared.

Tutte le cellule all&#39;interno del labirinto membranoso dell&#39;orecchio interno del mouse inclusi sensoriale, nonsensory ei neuroni gangliari a spirale sono tutti derivati ​​dallo placodally di derivazione otocyst adiacente al romboencefalo della ectoderma, intorno E8 10-14. Al E11, la regione ventrale del otocyst estende a formare il dotto cocleare e lo sviluppo continua, un gruppo di cellule epiteliali all&#39;interno della coclea, così come in altre regioni del otocyst, diventano specificato come ...

The mammalian organ of Corti is comprised of a mosaic of specialized cell types, including two types of mechanosensory hair cells as well as at least four types of nonsensory supporting cells making it an ideal model system to study normal cellular processes like proliferation, fate specification, differentiation and patterning. In addition, the normal development of these different cell types is essential for normal hearing function. Hence, it is crucial to understand the factors, both molecular and cellular, that regulate their development. However, the small size of the mouse cochlea as well as its inaccessibility poses a particular challenge for gene expression studies. Moreover, most of the cell fate specification and patterning events occur during embryonic time periods and are mostly completed before birth. Therefore, identification and characterization of signaling events during embryonic time periods is essential to gain insight into the molecular basis of cochlear morphogenesis. 
Here, we demonstrate a method to culture intact cochlea in vitro from embryonic mouse inner ears. The rationale behind the use of this technique is that cultured cochleae maintain their molecular and morphological characteristics thereby providing a valuable model for investigating potential candidate genes and exploring the mechanisms involved in cochlear morphogenesis. Although transgenic mice can be used for gene expression studies, an in vitro system is often needed for monitoring specific gene functions. Moreover, cochlear cultures can be established from transgenic mouse embryos so that their in vitro development and response to various soluble factors and antagonists can be studied. Although embryos at day 13 (E13) are used in this protocol, cultures from E12 or E14 to early postnatal inner ears can give similar results. 
We also present a gene transfer technique in cultured embryonic cochlear explants using square wave electroporation. Following the isolation of the cochlear explants, electroporation can be used to express DNA plasmids of gene(s) of interest in individual cells within the cochlear duct. This technique serves as a complementary approach to studies utilizing transgenic mice to gain insight into the molecular pathways underlying cellular phenotype. Using this method of gene transfer, a variety of epithelial cell types within the embryonic cochlea are transfected, thereby enabling loss-and gain-of-function analyses at the single-cell level. In addition, electrophysiological studies can also be performed in cochlear explant cultures8. This method of in vitro electroporation is relatively simple and straightforward, combined with minimal damage to the tissue, has resulted in a rapid expansion of this technique.

<table><tbody><tr><td>HBSS</td><td>Gibco</td><td>14065-056</td><td></td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Medium</td><td>Gibco</td><td>12430-054</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>10082</td><td></td></tr><tr><td>N-2 Supplement (100x)</td><td>Gibco</td><td>17502-048</td><td></td></tr><tr><td>Ciprofloxacin hydrochloride</td><td>Cellgro</td><td>61-277-RF</td><td></td></tr><tr><td>Glass Dish 60 mm</td><td>Kimble Chase</td><td>23062-6015/23064-6015</td><td></td></tr><tr><td>Glass Dish 100 mm</td><td>Kimble Chase</td><td>23064-10015/23062-10015</td><td></td></tr><tr><td>Minutien pins</td><td>Fine Science Tools</td><td>26002-15</td><td></td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools</td><td>11251-10</td><td></td></tr><tr><td>Pulse generator</td><td>Protech International Inc</td><td>CUY21Vivo-SQ</td><td></td></tr><tr><td>Glass bottom culture dishes</td><td>MatTek</td><td>P35G-0-10-C</td><td></td></tr><tr><td>Matrigel matrix</td><td>BD Biosciences</td><td>356237</td><td></td></tr><tr><td>Culture dish, 60 x 15 mm</td><td>Becton Dickinson</td><td>353037</td><td></td></tr><tr><td>Tissue culture dishes</td><td>Greiner Bio-one</td><td>639160</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>OS-30</td><td>Dow Corning</td><td>4021768</td><td></td></tr><tr><td>Fluoromount</td><td>Southern Biotech</td><td>0100-01</td><td></td></tr><tr><td>Conical tubes, 15 ml</td><td>Greiner Bio-one</td><td>188261</td><td></td></tr><tr><td>Myosin 6</td><td>Proteus Biosciences Inc</td><td>25-6791</td><td></td></tr><tr><td>Myosin 7a</td><td>Proteus Biosciences Inc</td><td>25-6790</td><td></td></tr><tr><td>TuJ1</td><td>Sigma</td><td>T2200</td><td></td></tr></tbody></table>

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 describe a method to isolate cochlea from embryonic inner ears and micro-dissect to expose the sensory epithelium. Once dissected, it may be plated and cultured as an intact cochlear duct (Figure 3) and analyzed by immunohistochemistry. The cultured cochlear explants provide a useful assay to examine the effect of variety of soluble factors and pharmacological drugs on cochlear development. Following dissection of cochlear explants, electroporation technique can be used to misexpress genes of interest...

Watch this Scientific Journal Video about Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation at JoVE.com

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

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.

Cultura della embrionali di topo cocleari espianti e Gene Transfer con elettroporazione

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

culture-embryonic-mouse-cochlear-explants-gene-transfer

Research

JoVE Journal

Developmental Biology

12.8K Views.  Medical University of South Carolina, College of Medicine. 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. 

Video: Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

Metodi di trasferimento Proprio localizzata di cellule o DNA in precoce post-impianto di embrioni di topo

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Ricerca

Biologia dello sviluppo

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.

Gene Transfer nel pollo Auditory Organ<em&gt; In Ovo</em&gt; Micro-elettroporazione

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 come un approccio chirurgico alla consegna di droga locale nelle orecchie interno dei topi adulti e neonati

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

Genetica

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 elettroporazione in vivo di Retina topo in via di sviluppo

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Neuroscienze

Gene Transfer to the Developing Mouse Inner Ear by In Vivo 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 the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

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

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

Gene Transfer per l&#39;orecchio del mouse Sviluppo Inner<em&gt; In Vivo</em&gt; Elettroporazione

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 ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

Cultura primaria e elettroporazione plasmidi dell&#39;Organo murini di Corti.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Biologia

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

A lungo termine Time Lapse Imaging del mouse cocleari Espianti

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

An enhanced platform for whole-embryo culture allows continuous and robust ex utero development of postimplantation mouse embryos for up to six days, from pregastrulation stages until advanced organogenesis. In this protocol, we detail the standard procedure for successful embryo culture using static plates and rotating bottle systems.

Ex Utero Coltura di embrioni di topo dalla pregastrulazione all'organogenesi avanzata

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. ...

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.

Metodi Ovo ed Ex Ovo per studiare lo sviluppo dell'orecchio interno aviario

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 ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

Neonatal Murine Cochlear Explant Technique come un<em&gt; In vitro</em&gt; Strumento di screening nella ricerca acustica

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Cultura della embrionali di topo cocleari espianti e Gene Transfer con elettroporazione

Riepilogo

Esplora Altri Video

Capitoli in questo video

Metodi di trasferimento Proprio localizzata di cellule o DNA in precoce post-impianto di embrioni di topo

Gene Transfer nel pollo Auditory Organ

Canalostomy come un approccio chirurgico alla consegna di droga locale nelle orecchie interno dei topi adulti e neonati

In elettroporazione in vivo di Retina topo in via di sviluppo

Gene Transfer per l'orecchio del mouse Sviluppo Inner

Cultura primaria e elettroporazione plasmidi dell'Organo murini di Corti.

A lungo termine Time Lapse Imaging del mouse cocleari Espianti

Ex Utero Coltura di embrioni di topo dalla pregastrulazione all'organogenesi avanzata

Metodi Ovo ed Ex Ovo per studiare lo sviluppo dell'orecchio interno aviario

Neonatal Murine Cochlear Explant Technique come un