Este trabalho foi financiado pela RIKEN Cérebro Science Institute (BSI), Human Frontier Science Program (HFSP) (atribuído ao TS) e Programa de Pesquisa Riken Júnior Associado (JRA, concedido a MK) RIKEN Cérebro Science Institute (BSI), Human Frontier Science Program (HFSP) (atribuído ao TS) e RIKEN Júnior Programa de Pesquisador Associado (JRA, concedido a MK) financiou este trabalho.

1. Preparação da solução capilar e DNA para injecção <ol><li> Purificar DNA plasmídeo com um Maxi-prep ou método equivalente, e fazer uma concentração final de 2-3 mcg / mL. Alíquota de 100 mg de DNA, adicionar 10μl do corante verde rápido (1% de ações) e TE (10 mM Tris Base, 1mM EDTA Solution, pH 8.0) e ajustar o volume para 100 ml para fazer a concentração final para DNA mix de injeção para 1μg / mL. A concentração de DNA plasmidial podem ser alteradas depende do tamanho do plasmídeo, e também a sua eficiência de transfecção, que precisa ser testado individualmente. </li><li> Girar a solução de DNA por 5 minutos a 14.000 RPM à temperatura ambiente e recolher o sobrenadante para remover qualquer resíduo. </li><li> Puxar um capilar de vidro tubos de vidro tubea capilar utilizando um extrator de micropipeta. Belisque a ponta com uma pinça para fazer a ponta tem um diâmetro M 20-30. Medida 800 M-1 mm da ponta e verifique o diâmetro externo é menor que 60 mm (Fig. 1A). </li><li> Conectar o capilar ao micromanipulador, e encha com óleo mineral. Para preencher o capilar com a solução de DNA, mergulhe a ponta na solução e sugar até a solução de DNA. </li></ol> 2. Vara eletrodos de platina Aqui usamos eletrodos revestidos de platina (Nepe gene, CUY611P2-1) para electroporate em região superficial do cérebro, como o córtex (Fig. 1F). 3. Preparação de micro-eletrodos Use micro-eletrodos para electroporated em partes profundas do cérebro (ie, o tálamo eo hipotálamo) (Fig. 2B-E). <ol><li> Uma extremidade do tungstênio (para eletrodos negativos) e platina (para eletrodos positivos) é enrolado em fios dourados e os pinos fixados com parafilme. Insira o fio à haste de um cotonete e embrulhar ambas as extremidades com parafilme (Figura 1B). </li><li> Uniformemente aguçar a ponta do fio com lixa até atingir 20-30 mM diâmetro externo e 60 mm de 800 M-1 mm da ponta (Fig. 1C e D). </li><li> Aplique uma camada fina de unha polonês para o fio para o isolamento. Após a unha polonês secar, use um cotonete embebido em acetona para removê-lo da ponta (cerca de 200 mM da ponta). Importante: A fim de evitar danos ao útero do rato, a parte dentro de 800 M-1 mm da ponta não deve ter mais de 70 m de diâmetro (Fig. 1E). </li></ol> 4. Preparação da cirurgia <ol><li> Anestesiar um rato grávidas (E9.5-E15.5). Demonstramos anestesiados com pentobarbital sódico grau farmacêutico (Tabela II, 50 mg por grama de peso corporal, injetar intraperitonealmente e aguarde 5 min). Alternativas para pentobarbital sódico como um anestésico Ketamina incluem injeção / Xilazina ou anestésicos de gás (gás é preferível para os procedimentos de curta duração). A injecção deve ser feito com atenção para evitar quaisquer órgãos, caso contrário ele irá causar danos para o útero ou matar animais. Quando IP sucesso é feito, a taxa de sobrevivência é mais do que 95%. Também a cirurgia pode ser realizada ao ar livre no meio ambiente. Antes de iniciada a operação, teste para a falta de resposta do animal a beliscar seus pés para confirmar anestésico. </li><li> Raspar o cabelo do abdômen com uma lâmina de barbear e EtOH 50%. Prepare a pele com a alternância de scrubs de betadine e álcool (70%). </li><li> Faça uma incisão na linha média abdominal com uma tesoura fina e retire todos os cornos uterinos com cuidado sobre a 37 ° C pré-aquecido tampão fosfato (PBS)-umedecidos gaze de algodão, que é colocado em torno da ferida. Manter a umidade do útero com PBS de todos os tempos. </li><li> Segure um cabo de fibra óptica flexível entre os dedos indicador e médio e colocá-lo sob o corno uterino. Cabo limpo com EtOH 70% antes do uso. Sem microscópio ou lupa é necessário para visualização. Útero posição entre o cabo de fibra óptica a luz óptica eo polegar, e aperte suavemente para empurrar para cima o embrião mais perto da parede uterina. </li></ol> 5. Injeção de DNA e eletroporação <ol><li> Quando embrião é posicionado, insira capilar de vidro cuidadosamente em ventrículo-alvo e injetar cerca de 1 mL de solução de DNA (Fig. 2A). </li><li> Eletroporação para a parte superficial da brainIf usando com eletrodos revestidos de platina para electoporation, coloque os eletrodos fora do útero e aplicar sugerido de onda quadrada pulsos de corrente de acordo com a instrução de fabricação (Tabela I). Se usingFor eletroporação a parte mais profunda do cérebro, eletroporação nmicro, eletrodos tipo eedle são usados. Iinsert um eletrodo de tungstênio fino negativo em DNA injetado ventrículo e um eletrodo de platina para o útero e região-alvo lugar entre dois eletrodos, em seguida, aplicar sugerido de onda quadrada pulsos de corrente (Tabela I) (Fig. 2B). </li></ol> 6. Eletroporação pós <ol><li> Coloque o corno uterino de volta em sua origlocalização inal com e adicionar 500 mL de 37 ° C pré-aquecido PBS. </li><li> Sutura a camada interna com sutura cirúrgica e camada externa estreita com um clipe autoclipauto 9 mm. </li><li> Animais colocar em uma almofada de aquecimento por 2 horas para permitir a recuperação da anestesia. Analgésicos como o carprofeno ou buprenorfina pode ser administrada para a dor pós-operatória. </li></ol> 7. Resultados representativos: Alguns exemplos de eletroporação cortical de pCAG-EYFP construir usando eletrodos de platina vara em momentos diferentes são mostradas na figura Figura 32. Cérebros são electroporated na E13.5, E14.5 e E15.5, e colhida no dia pós-natal (P) 6. ÅO coloração ntibody de RORc revela a posição da camada 4 e posição das células transfectadas EYFP são revelados por GFP coloração de anticorpos, e ambas as imagens são super impostas (Fig. 3A e B). Rotulados células corticais são camada de células corticais electroporated é claramente diferente em cada experimento (Fig. 32), o que sugere dependente do tempo eletroporação faz cortical camada específica GOF / LOF possível. A viabilidade de embriões e eficiência de transfecção de células varia dependendo da idade do embrião e do local de eletroporação. Amostras estão apresentadas na Tabela 1, no entanto, as condições devem ser otimizados para cada eletroporação dependendo do estágio e parte do cérebro. Eletroporação em tecidos profundos, tais como tálamo eo hipotálamo com o uso de eletrodos de platina pau muitas vezes dar baixa eficiência (Tabela 1). Este problema pode ser resolvido usando micro-eletrodos whichelectrodes, que chegará às partes profundas do cérebro. Figura 3 4 exemplo mostra de pCAG-EYFP eletroporação no diencéfalo desenvolvimento. Solução de DNA plasmídeo é injetado no 3 º ventrículo na E11.5 e seguido por micro eletroporação (Fig. 3C4C). A área é restrita electroporated no tálamo, onde a maioria do projeto axônios para o córtex (Fig. 3B4B). Axon projeção para camada 4 do córtex também pode ser visualizado por EYFP, que fillesfills o neurônio inteiro. (Fig. 3A4A). <img src="/files/ftp_upload/3024/3024fig1.jpg" alt="Figura 1"/> Figura 1. Esquemática dos desenhos animados demonstrando a forma correta do capilar. (A) Um tubo capilar de vidro é puxado através de um puxador de micropipeta para fazer certa forma. Uma dica é off prensado por uma pinça para torná-lo 20-30 mM. Medida 800 M-1 milímetro da ponta (faixa vermelha) e certifique-se que o diâmetro externo é menos de 60 mm (faixa verde). (B) end Um dos fios de tungstênio ou platina é enrolada em pinos banhados a ouro e fixado com parafilme. Em seguida, use uma lixa para fazer sua ponta para se tornar 20-30 mM diâmetro externo e 60 mm a partir de 1 mm da ponta. (C) Imagem do fio de tungstênio antes shaping. (D) Imagem do fio de tungstênio após a modelagem. (E) Imagem do fio de tungstênio após a aplicação de fina camada de unha polonês. Barra de escala em E é de 10 mM (menor escala) para CE. (F) Stick eletrodos de platina (Nepa gene. CUY611P2-1). Barra de escala em F é de 2 mm. <img src="/files/ftp_upload/3024/3024fig2.jpg" alt="Figura 2"/> Figura esquema dos desenhos animados 2. Procedimento cirúrgico. (A) Diagrama de injecção ventrículo lateral ou embriões de grandes dimensões (lado esquerdo) e em 3 º ventrículo ou embriões pequenos (lado direito). (B) Diagrama de electrporation pelo eletrodo de platina stick (lado esquerdo) e com agulha de eletrodo tipo (lado direito). <img src="/files/ftp_upload/3024/3024fig3.jpg" alt="Figura 3"/> Figura 3. Desenvolvimento do mouse telencéfalo foi electroporated com pCAG-EYFP no E13.5 (A), E14.5 (B) e E15.5 (C) e colhidas em P6. Cérebros foram seccionados coronal e corados com GFP anticorpo ou anticorpos RORc separadamente e imagens são mesclados. (A) de Eletroporação pCAG-EYFP plasmídeo em E13.5 transfectar muitos camada 4 neurônios. que se sobrepõem a camada de 4 marcadores RORc. (B) de Eletroporação pCAG-EYFP plasmídeo de células E14.5 transfecção, que são mais superficiais do que os neurônios da camada 4. (C) E15.5 eletroporação rótulo muitos neurônios superficial, como camada 2 / 3. Barra de escala 200 mM. <img src="/files/ftp_upload/3024/3024fig4.jpg" alt="Figura 4"/> Figura 4. Transfection da EYFP em tálamo-cortical axônios (TCA). (A) terminus TCA na camada cortical 4 é revelado por GFP coloração de anticorpos. RORc coloração do anticorpo é usado para revelar uma posição de camada 4. Ambas as imagens são tiradas de mesma seção e se fundiram. (B) Posição da eletroporação para o tálamo também é indicado pelo GFP coloração de anticorpos e coloração RORc, que a expressão é restrita no tálamo 11. (C) esquema dos desenhos animados para a eletroporação talâmica e projeção TCA são mostrados. Barra de escala 200 mM.

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	<li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation:+Visualization+of+neuronal+migration+in+the+developing+cortex.">Efficient in utero gene transfer system to the developing mouse brain using electroporation: Visualization of neuronal migration in the developing cortex.</a> Neuroscience. 103, 865-872 (2001).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Developmental Biology. 240, 237-246 (2001).</li><li>Fukuchi-Shimogori, T., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neocortex+patterning+by+the+secreted+signaling+molecule+FGF8.">Neocortex patterning by the secreted signaling molecule FGF8.</a> Science. 294, 1071-1074 (2001).</li><li>Kataoka, A., Shimogori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgf8+controls+regional+identity+in+the+developing+thalamus.">Fgf8 controls regional identity in the developing thalamus.</a> Development. 135, 2873-2881 (2008).</li><li>Imayoshi, I., Shimogori, T., Ohtsuka, T., Kageyama, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hes+genes+and+neurogenin+regulate+non-neural+versus+neural+fate+specification+in+the+dorsal+telencephalic+midline.">Hes genes and neurogenin regulate non-neural versus neural fate specification in the dorsal telencephalic midline.</a> Development. 135, 2531-2541 (2008).</li><li>Shimogori, T., Banuchi, V., Ng, H. Y., Strauss, J. B., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+signaling+centers+expressing+BMP,+WNT+and+FGF+proteins+interact+to+pattern+the+cerebral+cortex.">Embryonic signaling centers expressing BMP, WNT and FGF proteins interact to pattern the cerebral cortex.</a> Development. 131, 5639-5647 (2004).</li><li>Shimogori, T., Ogawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+application+with+in+utero+electroporation+in+mouse+embryonic+brain.">Gene application with in utero electroporation in mouse embryonic brain.</a> Development Growth &#38; Differentiation. 50, 499-506 (2008).</li><li>Takahashi, Y., Watanabe, T., Nakagawa, S., Kawakami, K., Sato, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avian+Embryology.">Avian Embryology.</a> Methods in Cell Biology. Vol 87, 271-271 (2008).</li><li>Feil, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand-activated+site-specific+recombination+in+mice.">Ligand-activated site-specific recombination in mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 93, 10887-10890 (1996).</li><li>Indra, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally-controlled+site-specific+mutagenesis+in+the+basal+layer+of+the+epidermis:+comparison+of+the+recombinase+activity+of+the+tamoxifen-inducible+Cre-ERT+and+Cre-ERT2+recombinases.">Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ERT and Cre-ERT2 recombinases.</a> Nucleic Acids Research. 27, 4324-4327 (1999).</li><li>Nakagawa, Y., O'Leary, D. D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+patterned+expression+of+orphan+nuclear+receptor+genes+ROR+alpha+and+ROR+beta+in+developing+mouse+forebrain.">Dynamic patterned expression of orphan nuclear receptor genes ROR alpha and ROR beta in developing mouse forebrain.</a> Developmental Neuroscience. 25, 234-244 (2003).</li></ol>

Não há conflitos de interesse declarados.

Neste protocolo, apenas descreveu os benefícios da técnica de transfecção pCAG-EYFP na área restrita de um cérebro pequeno embrionárias usando diferentes eletrodos em forma. Para comparação da eficiência de transfecção pelo promotor diferente é descrito anteriormente, o que mostra a especificidade de células do tipo 1. No entanto, existem limitações para a técnica, tais como a transfecção é apenas transitória e varia depender do plasmídeo, que não tem qualquer especificidade celular e é difícil controlar o número de células transfectadas. ThereforeHowever, combinando este protocolo com outras técnicas ainda mais possibilidades de fornecer métodos de manipulação diferentes. Primeiro, incorporando promotor célula específica no DNA plasmídeo permitirá célula do tipo de transfecção específicos, tais como neurônios, células gliais e astrócitos. Segundo, uma vez que a transfecção é transitória, não é adequado para cientistas que queiram analisar a função do gene mais tarde na vida. Portanto, a combinação com um transposase fazer DNA plasmídeo integrar o genoma irá convertê-lo em um transfecção estável 8. Adoção, ao lado do tet tet-on ou off-sistema, será possível manipular o tempo ou célula tipo específico de expressão gênica em células neurais 8. Alternativamente, pode-se combinar este protocolo com tamoxifeno induzível por Cre-ER (T) recombinases 9 e ratos repórter 10. Esta ampla acessibilidade dos tecidos vai mudar drasticamente projetos experimentais usados ​​em neurociência.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> diâmetro do fio desencapado de tungstênio 125 M </td><td> Sistemas AM </td><td> 797600 </td></tr><tr><td> diâmetro do fio desencapado de platina 125 mM </td><td> Sistemas AM </td><td> 767000 </td></tr><tr><td> Eletrodos revestidos </td><td> Nepa gene </td><td> CUY610P4-1 </td></tr><tr><td> tubo capilar de vidro </td><td> Stoelting </td><td> 50611 </td></tr><tr><td> micromanipulador </td><td> KD Scientific </td><td> KDS310 </td></tr><tr><td> tesoura </td><td> ROBOZ </td><td> RS-5865 </td></tr><tr><td> gerador de pulso </td><td> Sistemas de AM </td><td> Modelo 2100 </td></tr><tr><td> 9 milímetros autoclip </td><td> ROBOZ </td><td> RS-9260 </td></tr><tr><td> banhado a ouro pinos </td><td> WPI </td><td> 5482 </td></tr><tr><td> Anticorpo RORc </td><td> Perseus Proteomics </td><td> H3925 </td></tr></tbody></table>

mouse in utero electroporation: controlled spatiotemporal gene transfection

A fim de compreender a função de genes expressos em região específica do cérebro em desenvolvimento, incluindo moléculas de sinalização e orientação moléculas axônio, a transferência de genes local ou knock-out é necessária. Gene targeting knock-in knock-out ou em regiões local é possível realizar com a combinação com uma linha específica CRE, que é trabalhoso, caro e demorado. Portanto, um método de transfecção simples, uma em técnica de eletroporação útero, que pode ser realizada com curto período de tempo, será útil para testar a possível função de genes candidatos antes da geração de 1,2 animais transgênicos. Além disso, em eletroporação utero alvos áreas do cérebro onde nenhuma linha específica CRE existe, e vai limitar letalidade embrionária 3,4. Aqui, apresentamos um método de eletroporação no útero da combinação de dois diferentes tipos de eletrodos para a transferência de genes simples e conveniente em áreas-alvo do cérebro em desenvolvimento. Primeiro, um método de realização única de embriões utilizando um cabo de fibra óptica a luz óptica vai criar embriões pequenos (de E9.5) visível para injeção de DNA alvo em solução ventrículos e tipo de agulha de inserção de eletrodos para a área do cérebro alvo 5,6. A padronização do cérebro, tais como área cortical ocorrer na fase embrionária precoce, portanto, estes eletroporação início da E9.5 fazer uma grande contribuição para entender evento padronização toda área. Segundo, a forma precisa de um capilar evita danos uterina, fazendo buracos através da inserção do capilar. Além disso, a forma precisa dos eletrodos de agulha são criados com o tungstênio e fio de platina e afiada usando o papel da areia e isolados com unha polonês 7, um método que é descrito em detalhes neste protocolo. Esta técnica exclusiva permite a transfecção de DNA plasmidial em áreas restritas do cérebro e vai permitir embriões pequenos para serem electroporated. Isso vai ajudar a, abrir uma nova janela para muitos cientistas que estão trabalhando na diferenciação celular, migração celular, a orientação dos axônios em muito cedo estágio embrionário. Além disso, esta técnica permitirá aos cientistas transfecção de DNA plasmidial em partes profundas do cérebro em desenvolvimento como o tálamo eo hipotálamo, onde muitos não específicas da região linhas CRE existem para ganho de função (GOF) ou perda de função (LOF) analisa.

Watch this Scientific Journal Video about Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection at JoVE.com

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

Um método de transferência de genes no cérebro do rato em desenvolvimento é descrito usando um único método cirúrgico ea forma especial de eletrodos. Esta técnica exclusiva permite a transfecção de DNA plasmídeo temporal e espacialmente, o que ajudará muitos neurocientistas em estudar o desenvolvimento do cérebro.

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 ...

mouse-utero-electroporation-controlled-spatiotemporal-gene

Research

JoVE Journal

Neuroscience

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

42.7K visualizações.  RIKEN Brain Science Institute. Um método de transferência de genes no cérebro do rato em desenvolvimento é descrito usando um único método cirúrgico ea forma especial de eletrodos. Esta técnica exclusiva permite a transfecção de DNA plasmídeo temporal e espacialmente, o que ajudará muitos neurocientistas em estudar o desenvolvimento do cérebro.

Vídeo: Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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

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 ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

Entrega Gene eficiente em múltiplos territórios CNS Usando In Utero Eletroporação

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

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

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

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

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

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

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Visualização e manipulação genética de dendritos e espinhas no córtex cerebral e hipocampo do rato usando<em&gt; In utero</em&gt; Eletroporação

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

Geração de topicamente transgênicas Rats por<em&gt; In utero</em&gt; Eletroporação e<em&gt; In vivo</em&gt; Triagem Bioluminescência

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

Manipulação genética do rato em desenvolvimento Hipotálamo através<em&gt; In utero</em&gt; Eletroporação

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

Específicos do Striatum Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- e<em&gt; In Utero</em&gt; A electroporação em ratinhos C57BL / 6 mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

Imaginação confocal de lapso de tempo de neurônios migratórios na cultura de fatias organotípicas do cérebro de mouse embrionário Usando<em&gt; Em Utero</em&gt; Electroporação

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous functions of genes in the brain can be studied by inducing loss or gain of function in sparse populations of cells. Here, we describe in utero electroporation to deliver Cre recombinase into sparse populations of developing cortical neurons with floxed genes to cause loss of function in vivo.

Induzindo a recombinação de Cre-lox no córtex Cerebral de rato através da eletroporação no Utero

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...