Agradecemos ao Dr. Takahiko Matsuda para fornecer o pCAG-mCherry construir. YCM é suportado por um prêmio de Desenvolvimento de Carreira da Fundação Schweppe e uma bolsa da Fundação Whitehall.

1. Fornecimento Preparação (e não em vídeo) <ol><li> Prepare uma diluição fresco 1X de penicilina / estreptomicina (Pen / Strep) em 1X PBS. Não mais do que 50 mL é necessário. Filtrar com um filtro de seringa de 0,22 uM. </li><li> Preparar construções de injecção através da combinação de construções de plasmídeos desejados. Nós colocar todas as construções no vector pCAG-Flag com estequiometria adequada para um volume final de 10 ~ uL. Além disso, pCAG-GFP 14 ou pCAG-mCherry deve ser adicionado para a eficiência eletroporação rastreamento e células eletroporados. Usamos pCAG-mCherry para este experimento. Por fim, adicionar 0,22 m de filtrado corante Fast Green na mistura ADN plasmídico a uma concentração final de 0,05% para visualizar a eficiência injecção mesencefálicas bico embrionárias. </li></ol> 2. Preparação Egg (em vídeo) <ol><li> A fim de adquirir fase HH 11 embriões, óvulos precisam de ser incubada a 100 ° F (37,8 ° C) durante cerca de 41 horas após a fertilização com a humidade de 25-40%. Lugarovos do seu lado e marcar a parte superior de cada um com um traço de lápis. Coloque as bandejas em uma pré-aquecido, incubadora umidificada. Lentamente balançar ovos, definindo a função de giro de incubadora de plataformas para &quot;Automático&quot;. Verifique o nível de umidade e água a cada 24 horas. </li><li> Logo antes do início da experiência, remover os ovos da incubadora e deixar à temperatura ambiente. Isso vai ajudar a retardar o desenvolvimento e evitar o excesso de desenvolvimento. </li><li> Limpar a superfície de cada ovo com etanol a 70% e limpe para baixo com um Kimwipe. </li><li> Pegue dois pedaços de 4 cm de fita adesiva e colocá-los lado a lado sobre o ovo para fazer um retângulo grande sobre o traço do lápis. Alisar a fita contra o ovo. Este é o lugar onde a janela será feita. A fita irá impedir pequenos pedaços de casca de ovo de cair, quando a janela é cortado e aberto. </li><li> Usar uma seringa de 10 mL e 18 ½ agulha de calibre para desenhar 5 mL de albumina para fora do ovo. Após a perfuração da casca, o ângulo da agulha para longe da gema a não perturbarou danificar o embrião durante a remoção da albumina. Limpe a agulha com etanol 70% entre os ovos, mas certifique-se a agulha está seco antes de perfurar o próximo ovo. </li></ol> 3. Vidro Preparação agulha de injeção (em vídeo) <ol><li> As agulhas são feitas usando as seguintes configurações em um flamejante / Brown Micropipeta Puller (Sutter Instrument modelo P-97):. Calor 670, Pull 120, Vel 40, Hora 165 Mundial Precision Instruments (API) 4 polegadas 1,0 milímetros de vidro de parede fina capilar (API TW100F-4) é usado para fazer as agulhas. </li><li> Coloque uma agulha de vidro puxado com uma pipeta P10 ea ponta longa capilar carregamento pipeta (Eppendorf 5.242.956,003). Carregar uL 5-10, dependendo de quantas ovos são para ser injectada. Encher a agulha de vidro lentamente, criando uma coluna contínua de solução de plasmídeo ausente de bolhas de ar. </li><li> Coloque a agulha carregada no suporte da agulha ligado ao micromanipulador (Narishige MM3) ea Picospritzer III microinjector. Ver a agulha sob um dissectiem microscópio e aparar a agulha no ponto onde o fluido verde pára. Ela ajuda a usar um fundo escuro para visualizar melhor a agulha. Agulha apropriada corte vem com a prática. </li></ol> 4. Mesencéfalo / Injeção Tubo Neural (em vídeo) <ol><li> Use a tesoura para cortar uma mola janela 2-2,5 cm de diâmetro no ovo. Iniciar a janela do buraco da agulha anterior feito aquando da elaboração de albumina, e girar o ovo em sua mão enquanto você corta. Certifique-se de limpar a tesoura com um Kimwipe saturado em etanol 70% entre os ovos. </li><li> Ver embriões de galinha sob um microscópio de dissecção. Pode ser necessário para a formação dos olhos. Pode ajudar a visualizar o embrião injetando 0,22 m de filtrado 20% nanquim em PBS sob o embrião de galinha. No entanto, verificou-se que este passo deve ser evitada, se possível, uma vez que tende a diminuir a sobrevivência. </li><li> Injecção funciona melhor se o embrião está posicionado em paralelo para o caminho da agulha, com a cabeça para longe da agulha.Lentamente Pierce do tubo neural em um ângulo de 45 graus para a junção mesencéfalo rombencéfalo. É muito crítico para encher apenas a vesícula neural que representa o mesencéfalo com o DNA de plasmídeo e corante verde rápido, sem difusão significativa para as regiões do prosencéfalo e rombencéfalo. Iniciar injetar com o conjunto microinjector Picospritzer de duração de 25 ms, o que pode ter de ser alterado, dependendo da agulha de corte. Em geral, a utilização entre 10-50 ms. </li></ol> 5. Eletroporação (em vídeo) <ol><li> Recuperar a agulha de injeção. O local de 3 gotas de Pen 1X / Strep em solução de PBS no ovo no embrião. </li><li> Verifique as configurações do electroporator EMC830 BTX: <table border="1"><tbody><tr><td> TENSÃO: </td><td> 20 V </td></tr><tr><td> Comprimento do pulso: </td><td> 25 ms </td></tr><tr><td> PULSOS #: </td><td> 3 </td></tr><tr><td> INTERVALO: </td><td> 500 ms </td></tr><tr><td> POLARIDADE: </td><td&gt; UNIPOLAR </td></tr></tbody></table></li><li> Colocar os dois eléctrodos de platina mm de comprimento em forma de L ao mesmo nível horizontal como na parte inferior do tubo neural, com o mesencéfalo embrionário sentado no meio entre dois eléctrodos de 3 mm para além. O alinhamento dos eléctrodos com a parte inferior do tubo neural é crítica para a electroporação eficiente do mesencéfalo ventral. Para alcançar mesencéfalo específico eletroporação, que substituíram os eletrodos originais BTX com duas custom-made de platina em forma de L eletrodos com curtas 2 mm pontas longas. Estes eléctrodos de platina são feitos de fios de 0,5 mm de diâmetro de platina (Alfa Aesar). Estes eletrodos de platina são apenas o suficiente para cobrir o mesencéfalo de um embrião de galinha fase 11, que é de cerca de 0,9 mm de comprimento, sem visar a maior parte do cérebro anterior ou as regiões rombencéfalo. Pressione o pedal BTX uma vez. Bolhas de ar devem ser gerados em torno dos eletrodos com cada eletroporação sucesso. Retire cuidadosamente os eletrodos do ovo e limpe o electrodes fora com um Kimwipe 70% de etanol-coberto. Coloque mais 3 gotas de Pen 1X / solução Strep no ovo, no embrião. </li><li> Cobrir a janela com um 5 cm x 5 cm pedaço de fita de embalagem clara. Ter a certeza de selar o ovo bem e que os conteúdos do ovo não entram em contacto com a fita. </li></ol> 6. Incubar e Colheita <ol><li> Colocar os ovos de volta para a incubadora. Certifique-se que o giro da incubadora plataformas é desligada. Incubar ovos até que o desejado fase HH 23 é alcançada. </li><li> Embriões vivos colheita e colocá-los em pratos comuns PBS-cheias de Petri para cada condição. Tela de embriões colhidos sob um microscópio de dissecção fluorescente, mantendo apenas os embriões com expressão mCherry. </li><li> Transferência de embriões em poços individuais de uma placa de 24 poços. Encher cada poço com 1 mL de feita recentemente paraformaldeído a 2% (PFA) e permitir a fixação de 2-3 horas a 4 ° C. </li><li> Lavar embriões fixos três vezes durante 10 minutos cada em PBS. Coloque embriões seqüencialmente in 15% e 30% de sacarose até equilibrada. Os embriões serão inicialmente flutuar em sacarose, mas vai afundar em equilíbrio. </li><li> Verifique embriões sob um microscópio de dissecção fluorescente, tomando notas sobre o padrão de expressão mCherry eo nível de cada embrião. Encaixe cada embrião em outubro É muito crítico para embriões orientar correctamente para gerar secções do mesencéfalo óptimas cruzadas para análises subsequentes (Figura 1). </li><li> Preparar 18 uM secções do mesencéfalo de espessura usando uma Leica criostato CM3050S. Analisar o desenvolvimento do mesencéfalo e diferenciação de neurônios dopaminérgicos por imunocoloração com marcadores tipo adequado de células. </li></ol> 7. Os resultados representativos Uma representação esquemática de electroporating e analisando mesencéfalo pinto embrionário é mostrado na Figura 1. Em embriões de galinha com sucesso injetados e electroporados, expressão mCherry deve estar visível na região do mesencéfalo, após desenvolvimento ( <strong&gt; Figura 2A, B, C). Assumindo que mCherry se correlaciona com a expressão de outros co-eletroporados genes, os embriões que exibem expressão mCherry adequada pode então ser fixado e incorporado para preparar criossecções mesencéfalo para estudo posterior (Figura 2D). Geralmente, cerca de 50% de células progenitoras do mesencéfalo ventral neurais podem ser eficientemente transfectadas com o protocolo acima. A especificação do destino dopaminérgica no eletroporados progenitores neurais podem ser examinada pela co-localização de mCherry, Flag-etiquetados gene, com marcadores neuronais dopaminérgicos tais como tirosina hidroxilase (TH) (Figura 3). A padronização dos domínios progenitoras no mesencéfalo ventral pode ser investigado por co-immnostaining criosecções com marcadores de domínio diferentes progenitoras. Expressão mCherry insuficiente sugere que os genes ectópicos não são susceptíveis eficientemente expressa. Embriões sob esta condição não deve ser usado para estudos posteriores. Da mesma forma, os embriões quenão sobreviveu até ao fim do procedimento experimental não pode ser utilizado para a recolha de dados e análise. <img alt="A Figura 1" src="/files/ftp_upload/4017/4017fig1.jpg" /> Figura 1. Ilustração do mesencéfalo embrionário pinto no ensaio eletroporação in ovo. HH estágio 11 embriões de pinto embrionário são injectados com pCAG-mCherry juntamente com genes de interesse misturados com verde rápido para visualizar o processo de injecção (A). Depois de cheio com construções de ADN, mesencefálicas são electroporados com um electroporator de onda quadrada. Os embriões são ainda incubada até desejada fase HH 23 é alcançado. Em seguida, mCherry-positivas embriões são colhidas, fixadas e incorporado (B). Criossecções mesencéfalo são preparados com planos de corte indicados pela linha branca na Figura 1B, e analisados ​​por imunocoloração com marcadores do mesencéfalo, tais como tirosina hidroxilase (TH) (C). <a href="http://www.jove.com/files/ftp_upload/4017/4017fig1large.jpg" target="_blank">Clique aqui</a>para ver maior figura. <img alt="A Figura 2" src="/files/ftp_upload/4017/4017fig2.jpg" /> Figura 2. Preparação de criosecções de pinto mesencéfalo embrionário. Após 41 horas de incubação, a fase HH 23 embriões de galinha que expressam mCherry e os genes de interesse são colhidas (A, B e C), fixadas com PFA, tratadas com sacarose, e embebidos em OCT para cryosectioning. Embriões de galinha são cuidadosamente orientada para garantir que a preparação de secções do mesencéfalo. A morfologia do embrião, típica e do plano de corte para a obtenção de seções do mesencéfalo são mostradas (D). <a href="http://www.jove.com/files/ftp_upload/4017/4017fig2large.jpg" target="_blank">Clique aqui para ver maior figura</a> . <img alt="A Figura 3" src="/files/ftp_upload/4017/4017fig3.jpg" /> Figura 3. Análise de secções do mesencéfalo eletroporados. Criossecções a partir de embriões com níveis elevados de expressão mCherry (A) são preparadas e coradas com anticorposs que reconhecem a bandeira-etiquetados com gene de interesse (B) e mesencéfalo dopaminérgica neurónio marcador TH (C). Mesencefálicas embrionárias eletroporados com pCAG-mCherry e pCAG-Flag vetor vazio servir como controle. <a href="http://www.jove.com/files/ftp_upload/4017/4017fig3large.jpg" target="_blank">Clique aqui para ver maior figura</a> .

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Os autores não têm interesses conflitantes potenciais para divulgar.

A eletroporação em ovo de pinto mesencéfalo embrionário oferece um baixo custo e rápida alternativa para a geração de animais transgênicos ou knockout para realizar estudo in vivo de funções de genes no mesencéfalo de desenvolvimento neurônio dopaminérgico. Usando 2 mm curto eléctrodos de platina longos em forma de L, juntamente com a injecção de DNA embrionário mesencéfalo-específica é a chave para alcançar expressão eficiente do gene de interesse em progenitores do mese...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de Catálogo </td><td> Comentários </td></tr><tr><td> Ovos de galinha fertilizados </td><td> Charles River Laboratories </td><td></td><td></td></tr><tr><td> Ovo incubadora </td><td> GQF Manufacturing </td><td> 1502 Sportsman </td><td></td></tr><tr><td> BTX Electroporator </td><td> BTX Harvard Apparatus </td><td> ECM830 </td><td></td></tr><tr><td> Eletrodos </td><td> BTX Harvard Apparatus </td><td> 45-0162 em forma de L genetrodes </td><td> Para utilização com ECM830 electroporator </td></tr><tr><td> Irídio Platinum </td><td> Alfa Aesar </td><td> # 10056 </td><td> 0,5 mm de diâmetro Para fazer as 2 mm de comprimento eléctrodos </td></tr><tr><td> Tubo de vidro capilar </td><td> Instrumento de precisão Mundial </td><td> TW100F-4 </td><td></td></tr><tr><td> Microloader pipeta </td><td> Eppendorf </td><td> 5242 956.003 </td><td></td></tr><tr><td> Nanquim </td><td> Staples </td><td></td><td> Filtrar 20% antes da utilização </td></tr><tr><td> Microinjector </td><td> Automação Parker, Parker Hannifin Cooperação </td><td> Picospritzer III </td><td></td></tr><tr><td> Microscópio de dissecção fluorescente </td><td> Leica </td><td> MZ16F </td><td></td></tr><tr><td> Micropipeta extrator </td><td> Instrumento Sutter </td><td> D-97 </td><td></td></tr></tbody></table>

in ovo electroporation in chick midbrain for studying gene function in dopaminergic neuron development

Neurônios dopaminérgicos localizados no mesencéfalo ventral controle movimento, o comportamento emocional, e mecanismos de recompensa 1-3. A disfunção de ventral mesencéfalo dopaminérgica neurónios está implicado na doença de Parkinson, esquizofrenia, depressão, demência e 1-5. Assim, estudando a regulação da diferenciação de neurônios dopaminérgicos no mesencéfalo poderia não apenas fornecer informação importante sobre mecanismos de regulação do desenvolvimento de mesencéfalo e especificação destino neural progenitor, mas também ajudar a desenvolver novas estratégias terapêuticas para o tratamento de uma variedade de distúrbios neurológicos humanos. Neurônios dopaminérgicos diferenciar dos progenitores neurais que revestem a zona ventricular de mesencéfalo ventral do embrião. O desenvolvimento de células progenitoras neurais é controlada por programas de expressão génica 6,7. Aqui relatamos técnicas utilizando eletroporação para expressar genes especificamente no mesencéfalo de estágio Hamburger Hamilton (HH) 11 (thsomitos irteen, 42 horas) embriões de galinha 8,9. O desenvolvimento externo de embriões de galinha permite convenientes manipulações experimentais específicas em estágios embrionários, com os efeitos determinados em momentos posteriores de desenvolvimento 10-13. Tubos pintainho embrionárias neurais mais cedo do que HH fase 13 (dezanove sómitos, 48 ​​horas) consistem em multipotentes progenitores neurais que são capazes de se diferenciar em tipos de células distintas do sistema nervoso. O vector pCAG, que contém tanto um promotor de CMV e um pintainho β-actina potenciador, permite a expressão robusta de Flag ou outras construções de epítopo-etiquetados em tubos de bico embrionárias neurais 14. Neste relatório, destacamos medidas especiais para alcançar a expressão do gene regionalmente restrito em embrionárias mesencéfalo progenitores de neurônios dopaminérgicos, incluindo a forma de injectar DNA constrói especificamente para a região do mesencéfalo embrionário e como identificar eletroporação com pequenos feitos por eletrodos. Analisando cmesencéfalo caipira em fases posteriores proporciona um excelente sistema in vivo para plasmídeo vetor mediada por ganho de função e perda de função de estudos de desenvolvimento mesencéfalo. Modificação do sistema experimental pode prolongar o ensaio para outras partes do sistema nervoso para executar a análise de mapeamento de destino e para investigar a regulação da expressão do gene.

Watch this Scientific Journal Video about In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development at JoVE.com

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Para avaliar a função e regulação de genes durante o desenvolvimento de neurónios dopaminérgicos do mesencéfalo, nós descrevemos um método que envolve<em&gt; In ovo</em&gt; Electroporação de construções de DNA de plasmídeo em embrionárias de bico ventral do mesencéfalo progenitores de neurónios dopaminérgicos. Esta técnica pode ser utilizada para conseguir a expressão eficiente de genes de interesse para estudar diferentes aspectos do desenvolvimento mesencéfalo e diferenciação neuronal dopaminérgico.

Na eletroporação in ovo em Midbrain pintainho para estudar a função do gene em dopaminérgico Desenvolvimento Neuron

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

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In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

11.3K visualizações.  Children's Hospital of Chicago Research Center. Para avaliar a função e regulação de genes durante o desenvolvimento de neurónios dopaminérgicos do mesencéfalo, nós descrevemos um método que envolve In ovo Electroporação de construções de DNA de plasmídeo em embrionárias de bico ventral do mesencéfalo progenitores de neurónios dopaminérgicos. Esta técnica pode ser utilizada para conseguir a expressão eficiente de genes de interesse para estudar diferentes aspectos do desenvolvimento m...

Vídeo: In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

No útero Eletroporação seguido por Cultura Neuronal primário para estudar a função do gene no subconjunto dos neurônios corticais

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Neurociência

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

Aplicação de uma condutância do receptor NMDA em neurônios dopaminérgicos Rat Midbrain Usando a Técnica Clamp dinâmico

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Transfecção de células de camundongo por Ganglionares da Retina In vivo Eletroporação

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

Mouse No Utero Eletroporação: Transfecção Gênica Controlled Spatiotemporal

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Organotípicas culturas de mesencéfalo ventral Slice Embrionárias: Um Sistema para o Estudo de Desenvolvimento Neuronal dopaminérgicos In vitro</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

Em Expression in ovo de microRNA em Ventral Pintinho Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Dirigido neurônios dopaminérgicos Diferenciação de Células-Tronco pluripotentes humanas

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

Na imagem latente de cálcio Vivo de células de cabelo linha Lateral no Zebrafish Larval

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Studying Brain Function in Children Using Magnetoencephalography

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the human brain. MEG is quiet and less likely to induce claustrophobia compared with magnetic resonance imaging (MRI). It is therefore a promising tool for investigating brain function in young children. However, analysis of MEG data from pediatric populations is often complicated by head movement artefacts which arise as a consequence of the requirement for a spatially-fixed sensor array that is not affixed to the child's head. Minimizing head movements during MEG sessions can be particularly challenging as young children are often unable to remain still during experimental tasks. The protocol presented here aims to reduce head movement artefacts during pediatric MEG scanning. Prior to visiting the MEG laboratory, families are provided with resources that explain the MEG system and the experimental procedures in simple, accessible language. An MEG familiarization session is conducted during which children are acquainted with both the researchers and the MEG procedures. They are then trained to keep their head still whilst lying inside an MEG simulator. To help children feel at ease in the novel MEG environment, all of the procedures are explained through the narrative of a space mission. To minimize head movement due to restlessness, children are trained and assessed using fun and engaging experimental paradigms. In addition, children's residual head movement artefacts are compensated for during the data acquisition session using a real-time head movement tracking system. Implementing these child-friendly procedures is important for improving data quality, minimizing participant attrition rates in longitudinal studies, and ensuring that families have a positive research experience.

This article introduces a child-friendly research protocol designed to improve data quality by reducing head movement during pediatric magnetoencephalography (MEG). We familiarize families with the MEG environment, train children to remain still using an MEG simulator, and correct for residual head movement artefacts using a real-time head movement detection system.

Estudando a função cerebral em crianças usando magnetoencefalografia

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Análise Cinética 3D para a Avaliação Funcional no modelo de rat da lesão ciática de esmagamento nervoso

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...