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>

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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.

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			<td dir="LTR" height="43" style="height:43px;width:316px;">Dulbecco&#8217;s Modified Medium</td>
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			<td dir="LTR" style="width:161px;">12430-054</td>
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			<td dir="LTR" style="width:161px;">17502-048</td>
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			<td dir="LTR" style="width:161px;">23062-6015/23064-6015</td>
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			<td height="43" style="height:43px;width:316px;">Glass Dish 100 mm</td>
			<td dir="LTR">Kimble Chase</td>
			<td style="width:161px;">23064-10015/23062-10015</td>
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			<td height="43" style="height:43px;width:316px;">Minutien Pins</td>
			<td dir="LTR" style="width:192px;">Fine Science Tools</td>
			<td style="width:161px;">26002-15</td>
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			<td dir="LTR" style="width:192px;">Fine Science Tools</td>
			<td dir="LTR" style="width:161px;">11251-10</td>
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			<td dir="LTR">Protech International Inc</td>
			<td style="width:161px;">CUY21Vivo-SQ</td>
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			<td dir="LTR" height="43" style="height:43px;width:316px;">Glass Bottom Culture Dishes</td>
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			<td dir="LTR" style="width:161px;">P35G-0-10-C</td>
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			<td dir="LTR" height="43" style="height:43px;width:316px;">Matrigel Matrix</td>
			<td dir="LTR" style="width:192px;">BD Biosciences</td>
			<td dir="LTR" style="width:161px;">356237</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:316px;">Culture Dish, 60 X 15 mm</td>
			<td dir="LTR" style="width:192px;">Becton Dickinson</td>
			<td style="width:161px;">353037</td>
			<td></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:316px;">Tissue&#160; Culture Dishes</td>
			<td dir="LTR" style="width:192px;">Greiner Bio-one</td>
			<td style="width:161px;">639160</td>
			<td></td>
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		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:316px;">Phosphate Buffered Saline</td>
			<td dir="LTR" style="width:192px;">Gibco</td>
			<td dir="LTR" style="width:161px;">10010-023</td>
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			<td dir="LTR" height="43" style="height:43px;width:316px;">OS-30</td>
			<td dir="LTR" style="width:192px;">Dow Corning</td>
			<td dir="LTR" style="width:161px;">4021768</td>
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			<td dir="LTR" height="43" style="height:43px;width:316px;">Fluoromount</td>
			<td dir="LTR" style="width:192px;">Southern Biotech</td>
			<td dir="LTR" style="width:161px;">0100-01</td>
			<td style="width:167px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:316px;">Conical Tubes, 15ml</td>
			<td style="width:192px;">Greiner Bio-one</td>
			<td style="width:161px;">188261</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">Myosin 6</td>
			<td>Proteus Biosciences Inc</td>
			<td style="width:161px;">25-6791</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">Myosin 7a</td>
			<td style="width:192px;">Proteus Biosciences Inc</td>
			<td style="width:161px;">25-6790</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:316px;">TuJ1</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:161px;">T2200</td>
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		<tr height="43">
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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 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

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

Transferência de Gene eficiente em retinas pintainho para célula primária Estudos de Cultura: An<em&gt; Ex-ovo</em&gt; Eletroporação Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

Biologia do Desenvolvimento

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

Cultura e Co-Culture of Mouse ovários e folículos ovarianos

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

Ex Utero Eletroporação e Organotípicas Cultura Slice of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

Diferenciação Neural of Mouse Células-Tronco Embrionárias em soro livre de cultura em monocamada

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental manipulations in ovo/in vivo makes the chicken embryonic retina a versatile and very efficient experimental model. Although the chicken retina is easy to target in ovo by intraocular injections or electroporation, the effective and exact concentration of the reagents within the retina may be difficult to fully control. This may be due to variations of the exact injection site, leakage from the eye or uneven diffusion of the substances. Furthermore, the frequency of malformations and mortality after invasive manipulations such as electroporation is rather high.
This protocol describes an ex ovo technique for culturing whole retinal explants from chicken embryos and provides a method for controlled exposure of the retina to reagents. The protocol describes how to dissect, experimentally manipulate, and culture whole retinal explants from chicken embryos. The explants can be cultured for approximately 24 hr and be subjected to different manipulations such as electroporation. The major advantages are that the experiment is not dependent on the survival of the embryo and that the concentration of the introduced reagent can be varied and controlled in order to determine and optimize the effective concentration. Furthermore, the technique is rapid, cheap and together with its high experimental success rate, it ensures reproducible results. It should be emphasized that it serves as an excellent complement to experiments performed in ovo.

This protocol describes a method to dissect, experimentally manipulate and culture whole retinal explants from chicken embryos. The explant cultures are useful when high success rate, efficacy and reproducibility are needed to test the effects of plasmids for electroporation and/or reagent substances, i.e., enzymatic inhibitors.

Os explantes de retina inteiras de frango embriões para tratamentos de reagentes Eletroporação e Chemical

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental ...

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. ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

Cultura do Adulto Transgênicos Zebrafish Retinal explantes for Imaging Em células vivas, por Multiphoton Microscopia

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

Dissecção e cultura do rim embrionário do rato

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

Mediada por CRISPR perda de função de análise no grânulo cerebelar células usando no Utero Electroporation Gene transferência baseada

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Isolamento e diferenciação de Mioblasts primários de explantes do músculo esquelético do rato

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...