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: Todos os protocolos que utilizam animais vivos devem ser analisados ​​e aprovados por um cuidado e uso de animais Comitê Institucional (IACUC) e deve seguir métodos para o cuidado e uso de animais de laboratório oficialmente aprovado. Todas as dissecações deve ser realizada utilizando uma técnica estéril em um banco de fluxo laminar limpo. Luvas e máscara, se desejado, deve ser usado durante este procedimento. 1. Dissecção da Orelha Embryonic Rato Inner <ol><li> Configuração para órgão de Corti culturas de explantes: <ol><li> Esteriliza-se o fluxo laminar capuz de cultura de tecidos, ligando a luz UV durante cerca de 30 min e desinfectar a superfície com etanol a 70% antes da utilização. Além disso, esterilizar todos os instrumentos de dissecação, incluindo ferramentas de dissecação finos e pratos Sylgard-revestidos para dissecção embrião via autoclave ou por imersão em etanol 70% por pelo menos 20 min antes do uso. Deitar fora 70% de etanol e permitir que os pratos e instrumentos para ar seco em um fluxo laminar clea limpon bancada antes de usar. </li><li> Prepare a Solução Salina Equilibrada de Hank estéril (HBSS) / (4- (2-hidroxietil) -1-piperazinoetanossulfónico) (HEPES) mistura por adição de 10% de HBSS 10x e 0,5% de HEPES e ajustar o pH a aproximadamente 7,2 e esterilizar o filtro solução final e armazenar a 4 ° C. Realizar todas as dissecações em refrigerados solução HBSS / HEPES a melhor preservação do tecido durante todo o procedimento. </li><li> Pratos de vidro de fundo casaco com matriz da membrana basal por adição de 5 mL de água fria (4 ° C) estéril de Dulbecco Modified Eagle Médium (DMEM) a uma alíquota de 300 ul de matriz de membrana basal num tubo de polipropileno de 15 ml estéril. Misturar em vortex o conteúdo e adicionar 150 uL da mistura da matriz da membrana basal DMEM-a do centro de cada placa de cultura de modo a que cubra a totalidade da cultura assim presente no centro do prato. Cubra os pratos e armazenar em um 37 ° C incubadora por pelo menos 45 min antes do uso. </li><li> Despeje 5/4 ml refrigerados HBSS em three pratos de Petri de poliestireno e deixe fechado na capa. </li><li> Prepare meio de cultura num tubo de polipropileno de 15 ml estéril por adição de 9 ml de DMEM, 100 ul de suplementos de N2, 10 g / ml e Cipro 1 ml de FBS. </li></ol></li><li> O isolamento de embriões: NOTA: Neste procedimento para a cultura de explantes cocleares embrionárias de camundongos, ouvidos internos colheita do dia embrionário (E) 13 ossos temporais, com o dia após a fertilização sendo considerado como E1 9. O protocolo também pode ser utilizado com rato embrionário começando ouvidos internos de E12 a E18. <ol><li> Euthanize um rato cronometrado-grávida de CO 2 e sacrificar animais por deslocamento cervical ou protocolos aprovados adequados. Realizar eutanásia longe de de fluxo laminar de cultura de tecidos capuz para manter a esterilidade na sala cultura do tecido. </li><li> Coloque o mouse sacrificados em papel toalha com abdômen para cima e desinfectar abdômen com etanol 70%. </li><li> Abra a cavidade abdominal, agarrando pelecom uma pinça curva com uma mão e epiderme de corte e muscular ao longo da linha média com tesoura usando uma outra mão. Faça dois cortes perpendiculares em ambos os lados e retirar o útero cuidadosamente levantando cornos uterinos bilaterais com uma pinça e retirá-la da parte de baixo do tecido conjuntivo com uma tesoura. </li><li> Coloque a cadeia embrionárias em uma placa de Petri contendo solução refrigerada dissecção (mistura HBSS / HEPES) e transferir para o de fluxo laminar bancada limpa. </li><li> Remover cuidadosamente a partir de embriões placenta e colocá-los em uma das placas de Petri estéreis contendo solução de dissecção. Se a solução se torna sangrenta, movê-los para uma nova placa de Petri. NOTA: Neste ponto, os embriões podem ser encenado usando um guia de preparo Theiler. </li><li> Decapitar embriões por beliscar na região do pescoço com um par de fórceps limpas ou tesoura e coloque as cabeças em um prato fresco Petri contendo solução de dissecção gelada. </li></ol></li><li> Dissecção da orelha interna: <ol><li> Coloque ocabeça embrião ne em um prato Sylgard estéril contendo dissecação frio </li><li> solução. Trabalhando sob um microscópio de dissecação com uma ampliação de 1.6X ~, imobilizar a cabeça do embrião, colocando Minutien pinos em torno ou mais próxima da região dos olhos (Figura 1A). </li><li> Usando duas pinças estéreis cuidadosamente retire a pele e abrir o crânio no lado dorsal ao longo da linha média (Figura 1B). Remover cérebro de cavidade craniana e ouvidos internos localizados em ossos temporais podem ser identificadas nesta fase (Figura 1C, D). O forro do vaso sanguíneo, que normalmente está presente em torno das orelhas internas, pode ser utilizado como um ponto de referência para a identificação do ouvido interno, nesta fase (Figura 1D). </li><li> Dissecar o ouvido interno dos ossos temporais, colocando a pinça debaixo do tecido e isolamento do ouvido interno a partir da base do crânio e a transferência de um novo prato de dissecação contendo solução fria (Figura 1E, F). </li></ol></li></ol> 2. Geração de Órgão de Corti culturas de explantes NOTA: Nesta fase de desenvolvimento, o tecido cartilaginoso é e pode ser facilmente dissecados com a pinça. <ol><li> Oriente o ouvido interno para que o lado ventral está virada para cima (Figura 2A) e estabilizar o ouvido interno através da inserção suavemente a extremidade aguçada da Minutien pinos através da porção vestibular do ouvido interno (Figura 2B). </li><li> Usando fórceps ultrafinas cortar abrir a cartilagem sobrejacente, fazendo uma incisão utilizando uma ponta de pinça perto da janela oval e cuidadosamente remover a cartilagem da cóclea (Figura 2C). NOTA: É importante ter a certeza que os fórceps não são inseridos muito profundamente na cartilagem como cartilagem sobrejacente às vezes é fundido com o dueto coclear no caso em que é útil para utilizar uma pinça fechada para libertar cartilagem do ducto coclear subjacente por raspagem abaixo do surface da cartilagem. Nesta fase de desenvolvimento, a espiral da cóclea é de apenas três quartos de um turno de comprimento. </li><li> Em seguida, expor o epitélio sensorial, colocando a pinça na região preferida do ducto coclear, quer a base ou no vértice, e puxando para fora o telhado da cóclea (Figura 2D). NOTA: O telhado da cóclea deve ser removido completamente, uma vez que pode mascarar o epitélio sensorial impedindo a análise. NOTA: Este procedimento deve ser feito com cuidado, pois é fácil de rasgar o epitélio sensorial. </li><li> Como um último passo, cuidadosamente remover o tecido conjuntivo subjacente do epitélio sensorial exposta de modo que a base do explante coclear é uniformemente plana. </li><li> Isolar a cóclea dissecada da porção vestibular do ouvido interno com a pinça. Isto pode ser realizado antes ou após a etapa 2.4 (Figura 2E). </li><li> Transferir o epitélio sensorial da cóclea dissecado em uma membra porãopoço de cultura utilizando um cinzel estéril 1,5 milímetros (Figura 2F) ne-matriz revestida. </li><li> Oriente o explante da cóclea com a superfície luminal do epitélio virado para cima, e aspirar a solução de matriz de membrana basal DMEM para achatar cuidadosamente o tecido e substituí-la com 150 ul de meio de cultura fresco adicionando suavemente para o prato. Deve ser tomado cuidado para assegurar que cada explante adere bem à lamela de vidro revestido e não está a flutuar no meio de cultura. Certifique-se de que a ponta do fórceps, é apontado para cima, enquanto a fixação do poço de vidro para evitar danificar o explante da cóclea. </li><li> Suavemente transferir a placa de cultura estéril num prato de cultura de 150 milímetros e colocar numa incubadora de cultura de tecidos a 37 ° C com 5% de CO 2 durante período de tempo desejado, normalmente 3-6 dias in vitro (DIV). Depois de 1 DIV, examinar as culturas sob um microscópio de dissecação para certificar-se que o explante da cóclea aderiu bem à placa de cultura. </li><li> Depois de emcubation in vitro para comprimentos desejados de tempo, as culturas são processados ​​para imuno-histoquímica. NOTA: As culturas de explantes geralmente são incubadas durante 6 DIV, que é equivalente a fase de desenvolvimento, P0. Após a incubação in vitro, as culturas podem ser processados ​​em aplicações a jusante, como imuno-histoquímica (Figura 3B), RT-PCR, a hibridização in situ, e / ou Western blot. </li></ol> 3. A electroporação mediada por Transferência de Genes <ol><li> Configuração para eletroporação: <ol><li> Esterilizar um Sylgard 100 milímetros prato de vidro revestido por autoclavagem ou imersão em etanol a 70% durante cerca de 20 min e deixar secar ao ar numa bancada limpa antes da utilização. </li><li> Prepare o DNA de um vector de expressão de escolha utilizando Maximiliano ou midi-prep kits adequados. Os vectores de expressão utilizados neste protocolo são pIRES2-Atoh1.EGFP e pCLIG-NeuroD1.EGFP. A concentração final de ADN deve ser de pelo menos 1 ug / uL de ADN estéril /Livre de RNAse água. </li></ol></li><li> Electroporating ADN em explantes da cóclea embrionárias: <ol><li> Adicionar 10 ul de solução de ADN de plasmídeo para um novo Sylgard 100 milímetros revestida prato e transferir um explante da cóclea totalmente dissecados (obtido a partir do passo 2.5) para a solução de ADN. </li><li> Com a superfície luminal do epitélio virado para cima, a cóclea inclinar ligeiramente de modo que é perpendicular ao plano do prato. </li><li> Coloque as pás de eléctrodos em ambos os lados da cóclea com pá negativo (cátodo) para o epitélio sensorial e a pá positivo (ânodo) localizada adjacente à base da cóclea. Observação: Uma vez que o ADN tem carga negativa, que migra de negativo para positivo e do eléctrodo, colocando a extremidade do cátodo da sonda adjacente ao epitélio sensorial; o DNA principalmente ir através da superfície do epitélio sensorial. </li><li> Usando electroporator, entregar 9-10 pulsos de 24 mV, 30 ms duração do pulso com o interruptor do pedal em tele electroporator, e em seguida, adicione 100 ml de meio de cultura quente para a cóclea electroporado. </li><li> Repita os passos 3.2.1-4 para todos os explantes da cóclea e para o DNA de todos os genes de interesse e transferir as cócleas electroporadas na membrana basal revestida de matriz prato para o plaqueamento (passo 2.7). Cultura todas as cócleas electroporados em uma temperatura de 37 ° C, 5% de CO 2 incubadora humidificada para comprimentos de tempo desejados, que serão depois processadas para imunocitoquímica. NOTA: A cóclea de uma ninhada inteira de CD1 rato (aproximadamente 8-12 filhotes) pode ser isolado, microdissecados, electroporado e banhado em &lt;4 horas. </li></ol></li></ol> 4. Análise das Culturas Coclear explantes NOTA: As culturas são geralmente incubados durante 6 DIV, que é equivalente a fase de desenvolvimento, P0. Após a incubação in vitro, as culturas são fixadas e processadas para imunocitoquímica. <ol><li> Depois de comprimento desejado de incubation, remover o meio de cultura e lavar rapidamente o explante da cóclea em solução salina tamponada com fosfato (PBS) e incuba-se em paraformaldeído a 4% (em PBS) durante 15 min à temperatura ambiente. </li><li> Lavar o fixador por adição de PBS e lavar três vezes em PBS durante 15 min. </li><li> Permeabilizar utilizando PBS-T (PBS + Tween a 0,5%) durante 30 min antes de bloqueio com PBS-T contendo 10% de soro de cabra. </li><li> Incubar em solução de anticorpo primário contendo o anticorpo primário em PBS-T com 1% de soro de cabra durante a noite a 4 ° C. </li><li> Lava-se a cóclea três vezes à temperatura ambiente utilizando PBS-T 15 min antes da adição de cada anticorpos secundários conjugados com Alexa adequadas. </li><li> Uma vez que o explante é cultivado sobre coclear lamela de vidro que pode ser tratado individualmente pelo isolamento da lamela do prato de montagem e sobre a lâmina. Isto pode ser conseguido por imersão da placa de cultura em OS30 solvente para pelo menos 1 hora à temperatura ambiente. Usando a lâmina estéril, retire a lamela do dish e montar em um slide e visualizar em um microscópio fluorescente. </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.

Todas as células dentro do labirinto membranoso do ouvido interno de rato, incluindo sensorial, não-sensorial e os neurónios do gânglio da espiral são todos derivados de placodally derivado otocyst localizado adjacente à parte posterior do cérebro da ectoderme, cerca de 10-14 E8. Em E11, a região ventral do otocyst estende-se para formar o ducto coclear e como desenvolvimento continua, um grupo de células epiteliais no interior da cóclea, bem como em outras regiões do otocyst, tornar-se como emplas...

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...

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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 da embrionárias mouse cocleares explantes e Gene Transferência de Eletroporação

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 visualizações.  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. 

Vídeo: 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.

Métodos de Transferência Precisamente localizada de células ou DNA em adiantado postimplantation embriões de camundongos

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

Biologia do Desenvolvimento

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.

A transferência de genes para o frango auditivo órgão por<em&gt; In Ovo</em&gt; Micro-eletroporação

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 como uma abordagem cirúrgica para entrega de drogas para o ouvido interno de ratos adultos e Neonatal

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

Genética

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.

Em Eletroporação vivo de Retina do rato em desenvolvimento

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

Neurociência

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.

Transferência de genes para a orelha do camundongo Desenvolvimento Inner por<em&gt; In Vivo</em&gt; Eletroporação

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 primária e Eletroporação Plasmid do Órgão de Corti murino.

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.

Longo prazo Time Lapse Imagem do mouse cocleares explantes

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 Cultura de Embriões de Camundongos de Pregastrulation a Organogênese Avançada

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.

In Ovo e Ex Ovo Métodos para estudar o desenvolvimento do ouvido interno aviário

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.

Técnica de Explante Coclear Murino Neonatal como Ferramenta de Triagem In Vitro na Pesquisa Auditiva

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 da embrionárias mouse cocleares explantes e Gene Transferência de Eletroporação

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