This work was supported by grants from the Deutsche Forschungsgemeinschaft to SB (BR-2215; SFB 497/A9).

NOTA: Todas as experiências com animais foram realizados em conformidade com a lei alemã e foram aprovados pelos escritórios do governo em Tübingen. 1. Preparação de Micropipetas, Soluções e Membranas <ol><li> Preparação de micropipetas <ol><li> Puxe micropipetas de vidro usando um extrator micropipeta com o seguinte programa: Calor: 540, Pull: 125, Velocidade: 20 e Delay: 140. Os valores comprimento da agulha para 5,5 cm. </li><li> Agulhas cônicas usando um microgrinder para obter um tamanho adequado ponta de 4 mm. Armazene as agulhas em uma caixa ou 15 centímetros de Petri para evitar danos das dicas. </li></ol></li><li> Preparação de Soluções <ol><li> O plasmídeo de DNA Solução <ol><li> Preparar DNA de plasmídeo contendo a construção de ADNc desejado utilizando um kit Maxi-prep livre de endotoxina de acordo com o protocolo do fabricante. </li><li> Ajuste a solução de DNA do plasmídeo para uma concentração final de 3 mg / mL (GFP sem espigão vector) ou 4 ug /ul (com 1 ug de vector de GFP pico) em endotoxina libertar tampão Tris-EDTA contendo Fast Green (concentração final 0,05%). </li></ol></li><li> A laminina da solução <ol><li> Dissolve-se 1 mg de laminina em água estéril até um volume final de 1 ml. Prepare 100 mL alíquotas e armazenar a -80 ° C. </li></ol></li><li> A poli-L-lisina da Solução <ol><li> Dissolve-se 50 mg de poli-L-lisina em 50 ml de água estéril para uma concentração final de 1 mg / ml. Preparar alíquotas de 1 ml e loja em -20 ° C. </li></ol></li><li> Conclua solução salina equilibrada de Hank (HBSS completa) <ol><li> Prepare completa HBSS combinando 100 ml de HBSS 10x, 2,5 ml de 1 M de tampão HEPES (pH 7,4), 30 ml de 1 M de D-glucose, 10 ml de 100 mM CaCl 2, 10 ml de 100 mM de MgSO4, e 4 ml de 1 M de NaHCO3. Adicionar água estéril até 1 L e armazenar a 4 ° C. NOTA: Autoclave todas as soluções tampão de esperar de HEPES 1 M e 1 M de D-glicose, que são filter esterilizado. </li></ol></li><li> Slice Meio de Cultura <ol><li> Prepare fatia meio de cultura pela adição de 35 ml de Meio Basal de Eagle, 12,9 ml de HBSS completo (1.2.4), 1,35 ml de 1 M de D-glucose, 250 ul de 200 mM de L-glutamina, e 500 ul de penicilina-estreptomicina a obter um volume final de 50 ml. Adicionar soro de cavalo a uma concentração final de 5% e armazenar a 4 ° C. </li></ol></li><li> Low-Ponto de fusão (LMP) Agarose <ol><li> Prepara-se uma solução de agarose a 4% LMP por adição de 2 g de agarose LMP a 50 ml de HBSS completo (1.2.4), seguido por aquecimento num forno de microondas durante 1-2 minutos, a alta energia. Manter a solução num banho de água a 37-39 ° C. Guardar a solução a 4 ° C e reutilização. </li></ol></li><li> Solução paraformaldeído <ol><li> Numa hotte preparar uma solução a 4% de paraformaldeído (PFA) por adição de 4 g de paraformaldeído a 100 ml de PBS 1x. Aquece-se a solução para 60 ° C e adiciona-se algumas gotas de NaOH 1 N até que a solução se torna clear. </li></ol></li><li> Solução permeabilização <ol><li> Dissolve-se 9 g de BSA em 300 mL de PBS 1x contendo 0,3% Triton X-100 e armazenar a 4 ° C. Adicionar azeto de sódio a 10% para armazenamento a longo prazo. </li></ol></li></ol></li><li> Revestimento de Membrana inserções <ol><li> Dilui-se uma alíquota da solução de reserva de laminina (1.2.2) e uma aliquota de solução mãe de poli-L-lisina (1.2.3) em água estéril para um volume final de 12 ml. </li><li> Coloque inserções de membrana em placas de 6 poços com cada poço contendo 2 ml de água estéril. Adicionar 1 ml de solução de revestimento por cima da membrana e incubar O / N a 37 ° C numa atmosfera de 5% de CO2. </li><li> Após a incubação, lavar a membrana insere três vezes com 1 ml de água estéril e seco. Use pastilhas membrana revestida no mesmo dia ou armazenar a 4 ° C por até quatro semanas em um seco placa de 6 poços. </li></ol></li></ol> 2. Injecção de ADN e electroporação de embriões E15.5 e E18.5 <ol><li&gt; Anestesiar uma vez acoplado rato fêmea, colocando-a dentro de uma câmara de anestesia saturado com 5% de isoflurano e ligado a um vaporizador. Circule isoflurano e oxigênio a uma taxa de 1 L / min. Mantenha o animal na caixa de 2-4 min ou até inconsciente, que é testada por beliscar entre as patas do mouse. </li><li> Eutanásia o mouse inconsciente por deslocamento cervical no dia embrionário (E) 15,5 ou 18,5. Dissecar o útero contendo os embriões 13 e colocá-lo em uma placa de Petri contendo 15-20 ml de frio HBSS completa. NOTA: A partir deste ponto em diante, manter os embriões e tecidos no gelo. </li><li> Use um par de tesouras para separar cada embrião do corno uterino e lugar para uma segunda placa de Petri contendo frio HBSS completa. </li><li> Sob um microscópio de dissecação, cortar a parede do músculo uterino e da placenta, usando um par de fórceps finos (# 55) e tesoura. Solte cuidadosamente o embrião a partir do saco vitelino. </li><li> Use um par de tesouras de Bona para decapitate os embriões só acima dos membros anteriores em um ângulo de 60 °. Se a experiência requer genotipagem dos embriões, recolher uma amostra de tecido para isolamento de ADN genómico (um pequeno pedaço da cauda). </li><li> Transfira a cabeça para um prato limpo e seco Petri. Porque a cabeça tinha sido decapitado em um ângulo de 60 °, a cabeça deve inclinar para um lado quando colocado dorsal para cima. </li><li> Coloque uma agulha cuidadosamente para o meio do hemisfério perto da bregma (Figura 1A, B). Injectar cerca de 2-3 ul (em 3 ou 4 ug / uL) de solução de ADN por pisar o pedal do Picospritzer III usando 30 libras de pressão durante o período de 10-15 ms por impulso, aplicando impulsos de 5-8. A duração e número dos impulsos depende do diâmetro da abertura de agulha com aberturas menores que precisam de mais tempo. O intervalo entre cada pulso corresponde a 1 seg. </li><li> Antes da colocação dos eletrodos, aplique algumas gotas de HBSS completa sobre a cabeça do embryo. Colocar os eléctrodos de tal maneira que o terminal de &quot;negativo&quot; é no mesmo lado que o ventrículo injectado e o eléctrodo &quot;positivo&quot;, no lado oposto do ventrículo injectado abaixo da orelha da cabeça do embrião (figura 1C, D). Aplicar 5 pulsos de 50 V. <ol><li> Use 3 eletrodos mm para 15,5 E e 5 mm para eletrodos E 18.5. </li></ol></li></ol> 3. Dissecção do Cérebro <ol><li> Após a electroporação, descasca a pele da cabeça, com a ajuda de um par de fórceps finos. Utilizando um par de tesouras de mola fazer uma pequena incisão no meio do cerebelo na linha média do crânio. </li><li> Insira a tesoura primavera na incisão e cortar longitudinalmente ao longo da sutura sagital. Retire o crânio e separar o cérebro do crânio usando uma pinça fina. Transferir todo o cérebro em 15-20 ml frio solução HBSS completa. </li><li> Enquanto isso, coloque 4% LMP agarose,mantida a 37-39 ° C num banho de água, para um molde de casca-de distância. </li><li> Leve o cérebro para fora do HBSS completa por uma pequena espátula colher e drenar o excesso de HBSS usando lenço de papel fino ou Kimwipes. </li><li> Coloque todo o cérebro suavemente para dentro da agarose e ajustar a sua posição com uma agulha fina. Mantenha o molde em gelo até a agarose é solidificada e o bloco é segmentado (por cortes coronais os bulbos olfatórios apontar para cima). </li></ol> 4. Vibratome Seccionamento e Cultura Slice <ol><li> Apare os blocos de agarose LMP e cola-los para o palco amostra usando &#39;super cola &quot;. Depois de a cola de transferência é seca a fase espécime para a bandeja de tampão de vibratome e encher com gelo frio completar HBSS até que o bloco está imerso na solução. NOTA: Esterilizar todas as superfícies de instrumentos e equipamentos com etanol a 70% antes do corte. </li><li> Prepare 250 mm seções vibratome grossas usando uma lâmina nova como seguida. <ol><li> Apare o wi blocoth as seguintes configurações; freqüência - 60 Hz, amplitude - 0,7 mm, velocidade de 16-18 mm / sec. Corte as seções que contêm o tecido desejado com as configurações acima em velocidade lenta (9 mm / seg). Iniciando o seccionamento do cérebro posterior, recolher 5-7 seções do cérebro. </li><li> Transfira as seções para uma placa de cultura de 6 bem limpo contendo 5 ml de gelo frio completar HBSS com a ajuda de uma espátula dobrado e manter em gelo até que todas as seções são coletados (Figura 1E). </li></ol></li><li> Humedecer a membrana de forma cruzada com 100 ul de HBSS completa antes de colocar as secções sobre a membrana, a fim de facilitar a orientação das secções. </li><li> Transfira as seções usando uma espátula dobrado sobre a membrana (pegar um canto da seção com uma pinça e puxe para a espátula e, em seguida, use a pinça para empurrar a seção sobre a membrana). Coloque até cinco seções em uma membrana e organizar por usando uma pinça (Figura 1F). Não sobreponha as seções com os outros. <ol><li> Tirar o excesso de HBSS fora da membrana utilizando uma pipeta. As membranas específicas utilizadas aqui estão ligados a uma armação e inserido na placa de cultura de tecido, o que permite que o tecido estar em contacto, mas não coberta pelo meio. </li></ol></li><li> Coloque as inserções de membrana numa placa de 6 poços contendo 1,8 ml de meio de cultura de fatia (1.2.5) (Figura 1G, H). Incubar a placa de cultura a 37 ° C com 5% de CO 2 por 11 ou 14 DIV DIV. Mudar metade do meio (0,9 ml) a cada segundo dia. NOTA: Nesta fase, adicionar reagentes como bromodeoxiuridina (BrdU; 10 mM concentração final) para a rotulagem proliferação de células para a mídia pela primeira 20 horas do tempo de cultura. </li></ol> 5. A fixação das Seções Seguido por Imunofluorescência Coloração <ol><li> Use uma lâmina de bisturi limpa e afiada para cortar e aparar as membranas, dependendo da orientação doas seções. </li><li> Transferem-se as secções em conjunto com a membrana de uma placa de 24 poços contendo 1 ml de 4% PFA (1.2.7). Incubam-se as secções durante 1 hora à t.a., seguido por 3 lavagens com 1x PBS durante 15 min cada. Incubar as secções O / N com uma solução de permeabilização a 4 ° C com agitação suave. </li><li> No dia seguinte, incubar as secções com anticorpos primários apropriados, diluídos em solução de permeabilização, S / N ou durante 48 horas a 4 ° C com agitação suave. </li><li> Lavam-se as secções 3 vezes durante 15 min com PBS 1x e incubar O / N a 4 ° C com os anticorpos secundários apropriados diluídos em solução de permeabilização. </li><li> Após a incubação com os anticorpos secundários, lavar uma vez as secções com 1x PBS, durante 15 minutos, seguido por coloração com DAPI durante 10 min. </li><li> Lave as seções 3 vezes com PBS 1x por 15 min cada, e transferir para lâminas de microscópio. Adicionar ImmunoMount e coloque delicadamente uma lamela em cima das seções. Secar as lâminas O / N a 4 ° C e seal com unha polonês. NOTA: Mantenha os slides sempre a 4 ° C. <ol><li> Analisar as culturas fatia por microscopia confocal (Figura 1-I). </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

O hipocampo tem uma função importante na aprendizagem e memória. O giro dentado é também uma das duas regiões do cérebro onde ocorre a neurogénese não só durante o desenvolvimento, mas também ao longo da idade adulta. Pós-natal e neurogênese adulta rendimentos do hipocampo de uma forma semelhante envolvendo muitos fatores comuns. Definir os mecanismos de regulação desses fatores vai ser muito útil para a compreensão das doenças neurodegenerativas, que por sua vez levará a novas terapias e medidas prev...

O hipocampo desempenha um papel importante na memória e aprendizagem, bem como o comportamento emocional. Uma função principal consiste na consolidação da memória de curto prazo em memória de longo prazo, que requer alta plasticidade do sistema nervoso. O giro denteado do hipocampo actua como primário para a porta de entrada de informação de entrada e é também uma das duas regiões do cérebro com a neurogénese contínuo ao longo da idade adulta 1,2. O desenvolvimento da estrutura do hipocampo ocorre durante a embriogénese tardia e particularmente durante os primeiros 3 a 4 semanas pós-natal 3. Durante o desenvolvimento inicial do giro denteado um pool de células-tronco é estabelecida necessário para pós-natal, bem como a neurogênese adulta 4. Neurônios em desenvolvimento passam por várias etapas, desde a célula-tronco por várias fases de células progenitoras para os imaturos e, finalmente, o neurônio maduro durante a pós-natal, bem como neurogênese adulta. Em diferentes estádios da neurogénese a expressão degenes específicos é necessária para permitir a maturação e integração de novos neurónios do hipocampo para o circuito 5,6. Usando mouse genética e análise de fenótipo por imuno-histoquímica, bem como métodos moleculares permitiu definir o padrão de expressão e função de muitos destes genes. Além análise microarray, bem como imunoprecipitação da cromatina (CHIP) forneceram informações sobre possíveis genes alvos diretos e indiretos 7,8. No entanto, ainda há muitas questões em aberto sobre os mecanismos de regulação do desenvolvimento do hipocampo, em particular o desenvolvimento do giro denteado. Para obter mais conhecimentos específicos como os genes são regulados de um sistema é necessário permitir que a manipulação de um pequeno número de células por jusante ou a sobre-regulação do gene de interesse e / ou dos seus genes-alvo e seguir o seu destino durante o desenvolvimento. No útero electroporação de shRNAs, cDNA de genes de interesse ou Cre recombinase oferece essa ferramenta. Para assegurar a presença do DNA desejado ou pequenos RNAs de plasmídeos de expressão deve ser utilizado para a electroporação. Esta abordagem é implementado com muito sucesso no estudo do desenvolvimento cortical 9,10, mas é uma abordagem mais difícil de examinar o desenvolvimento do giro denteado, devido à posição das estruturas do hipocampo em camadas mais profundas do cérebro. Eletroporação utero Ex seguido de cultura fatia organotípica é uma abordagem para contornar esse problema 11,12. Em contraste no útero não electroporação todo o embrião, mas apenas a cabeça é usada, permitindo, por conseguinte, para colocar os eléctrodos de um modo mais favorável para dirigir o shRNA / DNA para o hipocampo e giro dentado. Nosso grupo empregada com sucesso ex utero eletroporação para estudar o papel do fator de transcrição Bcl11b durante o desenvolvimento do giro denteado 8. Bcl11b tem um duplo papel no desenvolvimento do giro denteado por regulating proliferação de células progenitoras, bem como a diferenciação como foi demonstrado por imuno-histoquímica. Para definir ainda um mecanismo para o envolvimento Bcl11b nestes processos, os protocolos do grupo Polleux 11,12 foram ajustados para estudar o giro denteado, conforme descrito abaixo na secção de protocolo. Numa primeira abordagem, a questão foi abordada se Bcl11b está regulando celular diferenciação de células neuronais de forma autônoma. A segunda abordagem examinou se desmoplakin, um gene alvo direto de Bcl11b, é suficiente para recuperar o fenótipo Bcl11b.

<table border="0" cellpadding="0" cellspacing="0" width="271">
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		<tr height="106">
			<td height="106" style="height:106px;width:71px;">Name of Reagent/ Equipment</td>
			<td style="width:73px;">Company</td>
			<td style="width:63px;">Catalog Number</td>
			<td style="width:64px;">Comments/ Description</td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:71px;">Flaming/ Brown Micropipette Puller</td>
			<td style="width:73px;">Sutter Instruments Company (USA)</td>
			<td style="width:63px;">P-97</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:71px;">Fine Glass Pipettes</td>
			<td style="width:73px;">Warner Instruments</td>
			<td style="width:63px;">G100F-4</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:71px;">Microgrinder</td>
			<td style="width:73px;">Narishige, Japan</td>
			<td style="width:63px;">EG-44</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:71px;">Anesthetic Bracket unit</td>
			<td style="width:73px;">Harvard Apparatus</td>
			<td style="width:63px;">PY2 34-0412</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:71px;">Halovet Vaporizer</td>
			<td style="width:73px;">Harvard Apparatus</td>
			<td style="width:63px;">PY2 34-0398</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:71px;">Fluovac System</td>
			<td style="width:73px;">Harvard Apparatus</td>
			<td style="width:63px;">PY2 34-0387</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:71px;">IMS Fluosorber</td>
			<td style="width:73px;">Harvard Apparatus</td>
			<td style="width:63px;">PY2 34-0415</td>
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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.

A ablação do factor de transcrição Bcl11b provoca a diminuição da proliferação de células progenitoras e a diferenciação neuronal resultando numa redução do tamanho do giro denteado e o número de células. Além disso neurônios mutantes não conseguem integrar-se no circuito do hipocampo causando aprendizagem e memória comprometimento 8. Para responder a perguntas sobre o mecanismo de regulação (s) de Bcl11b nestes processos eletroporação ex utero foi empregado. <p class="jove...

Watch this Scientific Journal Video about Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue at JoVE.com

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 ...

ex-utero-electroporation-organotypic-slice-culture-mouse-hippocampal

Research

JoVE Journal

Developmental Biology

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

9.4K visualizações.  University of Ulm. 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.

Vídeo: Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific Cre models, deletion of a protein throughout a tissue or cell population often leads to complex phenotypes resulting from multiple interacting mechanisms. Determining whether a phenotype results from disruption of a cell autonomous mechanism, which is intrinsic to the cell in question, or from a non-cell autonomous mechanism, which would result from impairment of that cell&#8217;s environment, can be difficult to discern. To gain insight into protein function in an in vivo context, in utero electroporation (IUE) enables gene deletion in a small subset of cells within the developing cortex or some other selected brain region. IUE can be used to target specific brain areas, including the dorsal telencephalon, medial telencephalon, hippocampus, or ganglionic eminence. This facilitates observation of the consequences of cell autonomous gene deletion in the context of a healthy environment. The goal of this protocol is to show how IUE can be used to analyze a defect in radial migration in a floxed transgenic mouse line, with an emphasis on distinguishing between the cell autonomous and non-cell autonomous effects of protein deletion. By comparing the phenotype resulting from gene deletion within the entire cortex versus IUE-mediated gene deletion in a limited cell population, greater insight into protein function in brain development can be obtained than by using either technique in isolation.

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment.

Indução da Eliminação Proteína Através<em&gt; In Utero</em&gt; Eletroporação para definir défices de Migração Neuronal em modelos transgênicos

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific ...

Biologia do Desenvolvimento

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 ...

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 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 ...

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Cultures Organotípicas fatia de Estudos de Pós-natal Neurogênese

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

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 Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

Ex Cultura Vivo de Pintainho Cerebelar Slices e voltadas para o espaço Eletroporação do grânulo precursores de células

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

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 ...

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate embryonic origins and functions &#8212; the neurohypophysis and the adenohypophysis. The developing mouse pituitary gland is tiny and delicate with an elongated oval shape. A coronal section is preferred to display both the adenohypophysis and neurohypophysis in a single slice of the mouse pituitary.
The goal of this protocol is to achieve proper pituitary coronal sections with well-preserved tissue architectures from developing mice. In this protocol, we describe in detail how to dissect and process pituitary glands properly from developing mice. First, mice are fixed by transcardial perfusion of formaldehyde prior to dissection. Then three different dissecting techniques are applied to obtain intact pituitary glands depending on the age of mice. For fetal mice aged embryonic days (E) 17.5 - 18.5 and neonates up to 4 days, the entire sella regions including the sphenoid bone, gland, and trigeminal nerves are dissected. For pups aged postnatal days (P) 5 - 14, the pituitary glands connected with trigeminal nerves are dissected as a whole. For mice over 3 weeks old, the pituitary glands are carefully dissected free from the surrounding tissues. We also display how to embed the pituitary glands in a proper orientation by using the surrounding tissues as landmarks to obtain satisfying coronal sections. These methods are useful in analyzing histological and developmental features of pituitary glands in developing mice.

We present a protocol to dissect pituitary glands and prepare pituitary coronal sections from developing mice.

Dissecação e preparação Coronal fatia de desenvolver Mouse hipófise

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

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 ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

Cultura de organotypic método para estudar o desenvolvimento de tecidos embrionários de galinha

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Ex Utero Eletroporação e Organotípicas Cultura Slice of Mouse Hippocampal Tissue

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