Name: cortex-, hippocampus-, thalamus-, hypothalamus-, lateral septal nucleus- and striatum-specific in utero electroporation in the c57bl/6 mouse
Uploaded: 2016-01-18T14:00:00.000+00:00
Duration: 6 min
Description: Watch this Scientific Journal Video about Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse at JoVE.com

Technical supported by Melanie Pfeifer and Nikolai Schmarowski (Institute for Microscopic Anatomy and Neurobiology, University Medical Center Mainz).

Declaração de Ética: A manipulação dos ratos e os procedimentos experimentais foram conduzidos de acordo com as directrizes europeias, nacionais e institucionais para cuidados com os animais. 1. In Utero Eletroporação Nota: No útero electroporação foi realizada como publicado anteriormente 12,14. Portanto, o método só é descrito resumidamente a seguir (Figura 1). <ol><li> Preparativos <ol><li> Preparar a solução rápida de cor verde livre de endotoxina avançada plasmídeo transfecção-grade (contendo 1,5 pCAGGS), como descrito anteriormente 12. </li><li> Puxe e moer (35 ° de ângulo de capilares de vidro de borosilicato) (0,8-0,9 de diâmetro) para injectáveis. </li><li> Esterilizar instrumentos. </li></ol></li><li> Cirurgia <ol><li> Administrar analgésicos (carprofeno, 4 mg / kg de peso corporal, sc, 24 h) depósito, pelo menos, 30 min antes de iniciar a cirurgia. </li><li> Manter o comprimento dea anestesia para um máximo de 35-45 min (fase III - fase de anestesia cirúrgica de acordo com a Guedel 15 - máximo de 25-30 min). </li><li> Anestesiar ratos cronometrados grávidas com isoflurano (indução de uma câmara de: 2,8%, a cirurgia por máscara: 2,5%). Confirme anestesia por falta de resposta aos pés-pitada. </li><li> Aplicar pomada para evitar a secura ocular durante a anestesia. </li><li> Esterilizar a área cirúrgica com 7,5% de solução providona-Iodo e cobrir o mouse com gaze estéril (úmido com solução salina 0,9% benzilo álcool), com apenas a área cirúrgica exposta. </li><li> Cortar abrir a cavidade abdominal com um bisturi (incisão na pele: 1.5-2 cm, incisão muscular: 1-1,5 cm). </li><li> Preserve a cavidade abdominal aberta de secar umedecendo com solução salina aquecida álcool benzílico a 0,9% ou outra solução isotônica estéril como dirigido por pessoal veterinário ou comitê. </li><li> Extrair cuidadosamente cornos uterinos (usando uma pinça de anel, não toque no útero com o dedos). </li></ol></li><li> A injecção de ADN e electroporação. Nota: Os detalhes exatos para segmentar regiões específicas são indicadas na secção 2. <ol><li> Injectar-se lentamente a solução de cor de ADN para o ventrículo como indicado na Figura 1 e da secção 2 através dos capilares de borosilicato preparados. </li><li> Aplique a tensão adequada para a fase embrionária correspondente (por exemplo., 36-38 V para E14) através de eletrodos especializados pinças-platina (ciclo de intervalo de comprimento de 50 ms, intervalo de pausa 950 ms). As pás de electroporação de pulso durante a aplicação da tensão para minimizar os danos da parede uterina. </li></ol></li><li> Publicar eletroporação / cuidados pós-operatórios <ol><li> Recolocar cuidadosamente os cornos uterinos na cavidade abdominal. </li><li> Suturar o músculo e a incisão na pele separadamente. </li><li> Deixe o resto do mouse em um dispositivo de suporte térmico para a recuperação. </li><li> Supervisionar o mouse durante a recuperação (comportamento, alimentação econsumo de água), 4 horas, 24 horas e 48 horas após a cirurgia. Se necessário, aplicar analgésicos adicionais (carprofeno, 4 mg / kg de peso corporal, 24 horas depósito) para mais 2 dias. </li></ol></li></ol> 2. A transfecção de áreas cerebrais específicas Nota: A posição do pólo positivo é mostrado como o ângulo entre a linha de centro vertical através do ventrículo preenchido com a solução de ADN (o ventrículo direito ou terceira) e a posição do pólo positivo. Ao encher o ventrículo esquerdo as posições são invertidas-espelho. Na Figura 4 as posições da orelha primordial são mostrados, que fornecem importantes pontos de referência. <ol><li> A transfecção de áreas corticais (Figura 2A, a Tabela 1). <ol><li> Realizar a cirurgia, como descrito na seção 1.2. Para a electroporação em útero de áreas corticais utilizar embriões E14. </li><li> Injectar 1,5-2 ul de solução de ADN-ADN contendo 4 ug no ventrículo lateral direitoatravés dos capilares de borosilicato preparados. <ol><li> Posicione cuidadosamente o embrião usando uma pinça anel de modo que não há vasos visíveis acima do local da punção. </li><li> Injectar-se lentamente a solução de DNA de aproximadamente 0,75 mm coronal da sutura lambdoidal e 0,5 mm lateral da sutura sagital (Figura 3). Certifique-se que a profundidade de inserção é de 1,2 mm (E14, medido a partir da parede uterina). Verifique se há uma coloração azul-verde com uma demarcação nítida. </li></ol></li><li> Aplicar tensão através das pinças-eletrodos de platina especializados (ciclo de intervalo de comprimento de 50 ms, o intervalo de pausa 950 ms). <ol><li> Use um eletrodo de pá 3 ou 5 mm. </li><li> Utilize uma tensão que varia de 36-38 V. </li><li> Colocar o centro dos eléctrodos apenas anterior dos primórdios da orelha. </li><li> Para transfectar os ângulos de uso córtex retrosplï¿½icos 330-0 °, para transfectar o córtex motor 0-40 °, para transfectar o córtex somatossensorial 40-95 °, para transfectaro córtex piriforme 95-110 ° (Figura 2A). </li></ol></li><li> Execute pós eletroporação / cuidados pós-operatórios, como descrito na secção 1.4. Sacrificar os ratos e analisar os embriões de 4 dias após a eletroporação (E18) conforme descrito na seção 3. </li></ol></li><li> A transfecção do hipocampo (Figura 2B, Quadro 1). <ol><li> Realizar a cirurgia, como descrito na seção 1.2. Para a electroporação em útero de formação hipocampal utilizar embriões E15. </li><li> Injectar-2 uL de solução de ADN contendo 4 ug de ADN no ventrículo lateral direito através dos capilares de borosilicato preparados. </li><li> Aplicar tensão através das pinças-eletrodos de platina especializados (ciclo de intervalo de comprimento de 50 ms, o intervalo de pausa 950 ms). <ol><li> Use um eletrodo de pá 3 ou 5 mm. </li><li> Utilize uma tensão que varia de 38-40. </li><li> Colocar o centro do pólo negativo imediatamente anterior à orelha e o primórdio do centro Of o pólo positivo no meio de o primórdio da orelha. </li><li> Para transfectar as giro dentado usar ângulos de 190-210 °, para transfectar as Ammonis cornu (CA) 3 210-230 °, para transfectar as CA2 230-250 °, para transfectar as CA1 250-275 ° (Figura 2B). </li></ol></li><li> Execute pós eletroporação / cuidados pós-operatórios, como descrito na secção 1.4. Análise após a formação do hipocampo completo (após o nascimento, p21) executar conforme descrito na seção 3. </li></ol></li><li> A transfecção do núcleo septal lateral e corpo estriado (Figura 2C, Tabela 1) Nota: Os ângulos de sobreposição com aqueles para transfecção do hipocampo. Para transfectar eficazmente o núcleo septal lateral e o corpo estriado sem transfecção hipocampo indesejado fases anteriores embrionárias (E12) tem que ser usado. <ol><li> Realizar a cirurgia, como descrito na seção 1.2. Para a electroporação in utero do núcleo septal lateral e striathum usar embriões E12. </li><li> Injectar 1 ul de solução de ADN-contendo 4 ug de ADN no ventrículo lateral direito através dos capilares de borosilicato preparados. </li><li> Aplicar tensão através das pinças-eletrodos de platina especializados (ciclo de intervalo de comprimento de 50 ms, o intervalo de pausa 950 ms). <ol><li> Usar um eléctrodo de 0,5 mm. </li><li> Use 33 V. </li><li> Colocar o centro dos eléctrodos ao nível dos primórdios da orelha. </li><li> Para transfectar os ângulos de uso do núcleo septal lateral 100-170 °, para transfectar as estriado 170-240 ° (Figura 2C). </li></ol></li><li> Execute pós eletroporação / cuidados pós-operatórios, como descrito na secção 1.4. Sacrificar os ratos e analisar os embriões 6 dias após a eletroporação (E18) conforme descrito na seção 3. </li></ol></li><li> A transfecção do tálamo e hipotálamo (Figura 2D, Tabela 1) <ol><li> Realizar a cirurgia, como descrito na seção 1.2. Para o em Utero eletroporação do tálamo e hipotálamo uso de embriões E12-E13. </li><li> Injectar-1-1,5 ul de solução de ADN contendo 4 ug de ADN no ventrículo lateral (esquerda ou direita) através de capilares de borosilicato preparados. Espera durante 2-3 min, até a solução se difunde para o terceiro ventrículo. Alternativamente injectar directamente no terceiro ventrículo. Nota: Isso melhora especificidade, mas pode causar uma taxa de sobrevivência diminuiu (de alto risco de danos). </li><li> Aplicar tensão através das pinças-eletrodos de platina especializados (ciclo de intervalo de comprimento de 50 ms, o intervalo de pausa 950 ms). <ol><li> Usar um eléctrodo 0,5 ou 3 mm. </li><li> Utilize uma tensão que varia de 33-35 V. </li><li> Colocar o centro dos eléctrodos apenas posterior os primórdios da orelha. </li><li> Para transfectar os ângulos de utilização a partir de 25-40 ° tálamo, para transfectar o hipotálamo 90-180 ° (Figura 2D). </li></ol></li><li> Execute pós eletroporação / cuidados pós-operatórios como descrita no ponto 1.4. Sacrificar os ratos e analisar os embriões 6 dias após a eletroporação (E18) conforme descrito na seção 3. </li></ol></li></ol> 3. Perfusão Fixation <ol><li> Preparativos <ol><li> Quente 4% de paraformaldeído (PFA) a 37 ° C. </li><li> Encha uma seringa de 50 ml com PBS (4 ° C) e uma segunda seringa aquecido com PFA. Evite bolhas. </li><li> Conecte as seringas a uma torneira de três vias. Conecte o terceiro fim a um conjunto de infusão voada (borboleta). </li><li> Lave o conjunto de infusão voado com PBS. </li></ol></li><li> Perfusão <ol><li> Profundamente anestesiar os ratos (ou pós-natal grávida transfectadas) com aplicação subcutânea de cloridrato de cetamina (60 mg / kg) e xilazina (7,5 mg / kg). </li><li> Confirme o estágio de tolerância cirúrgico por falta de resposta aos pés-pitada. </li><li> Abra a cavidade abdominal logo abaixo da caixa torácica. </li><li> Corte cuidadosamente o diafragma. </li><li> Cuidadosamente abra a caixa torácica através de um corte na each local até a clavícula. </li><li> Inserir a ponta da borboleta para dentro da câmara do coração esquerdo (a partir do ápice do coração). </li><li> Corte o apêndice atrial esquerdo. </li><li> Lentamente perfundir o rato com PBS (aproximadamente 10 ml). </li><li> Alterne a torneira de três vias. </li><li> Perfundir o mouse com 5-10 ml PFA 4%. </li><li> Decapitar o mouse com uma tesoura e dissecar o cérebro começando no forame magno. Realize a dissecção como publicado anteriormente 16. </li><li> Postfixate o cérebro por 2 dias em PFA 4%. Manter os cérebros a 4 ° C no escuro. </li></ol></li></ol> 4. A imuno-histoquímica <ol><li> Gerar 70 um secções (por exemplo, com um vibratome). </li><li> Opcional: aumentar a fluorescência com coloração de anticorpos. <ol><li> Bloquear as secções durante 1 hora à temperatura ambiente (0,1-0,2% de Triton X-100, 5% de soro de cabra normal) </li><li> Incubar O / N com o anticorpo apropriado (anti-GFP, 1: 1000) </li><li> Lavar 3-5 Vezes com tampão de 0,1 M fosfato. </li><li> Incubar 3-5 h com o anticorpo secundário conjugado com fluorescência apropriado (Alexa Fluor 488 anti-coelho-IgG conjugada, 1: 1000). </li><li> Opcional: contracoloração com 4-6-diaminodino-2-fenilindole (DAPI, 0,2 g / ml em tampão fosfato 0,1 M) durante 1 min. </li><li> Analise de fluorescência com um microscópio de fluorescência apropriado. </li></ol></li></ol>

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The authors have nothing to disclose.

This protocol describes in detail how to transfect 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 of C75BL/6 mice. With all the provided information this is the first protocol, which supplies all necessary information to easily recreate transfections of these cerebral regions in the C57BL/6 mouse. Previous publications are mostly focused only on a few specific r...

Desde a primeira descrição em 2001 por três grupos independentes 1-3 i n útero electroporação tornou-se uma ferramenta padrão amplamente utilizado para analisar a expressão génica no sistema nervoso central de roedores. Em comparação com a geração de camundongos knockout, que é, apesar de técnicas de melhoria contínua, ainda que consome tempo e dinheiro, os apelos in utero eletroporação, devido à sua simplicidade. Então, in utero eletroporação permite gain- rápido e eficiente e perda de função estudos 4. Para transfectar as regiões cerebrais, a solução contendo o plasmídeo carregado negativamente é injectada num ventrículo. Durante o pulso eléctrico, o DNA carregado negativamente migra em direcção ao pólo positivo e, por conseguinte, a região transfectadas podem ser seleccionadas simplesmente alterando a posição do pólo positivo. Tem sido frequentemente demonstrado que várias regiões do sistema nervoso central pode ser TArgeted 3,5-8. Por exemplo, estudos recentes mostram transfecções específicos do hipocampo, córtex piriforme ou o estriado 9-11. No entanto, as informações sobre as posições adequadas são muitas vezes apenas mal padronizados e nem sempre são fáceis de transferir para diferentes linhagens de camundongos. A transfecção de certos estágios embrionários está longe de ser trivial. Muitos fatores influenciam deve ser levado em consideração ao escolher o set-up para específica in utero eletroporação. Em primeiro lugar, para transfectar de forma otimizada as respectivas fases embrionárias, é necessário o conhecimento sobre as tensões apropriadas. Altas tensões diminuir a taxa de sobrevivência, enquanto que baixas voltagens reduzem a eficiência de transfecção 2,3,12. Também o tamanho da pá eletrodo desempenha um papel crucial, porque o uso de pás de eletrodos que são demasiado grandes resultados em menor especificidade ou podem causar a morte devido ao carinho do ritmo cardíaco 4,12,13. O aplicadatensão e do tamanho e da posição do eletrodo de remo são as características mais importantes a considerar, mas também há outros fatores que influenciam o resultado da eletroporação, como a quantidade aplicada de solução de DNA. Nós desenvolvemos um protocolo detalhado que permite a transfecção rápido e eficiente de várias regiões do cérebro de C57BL / 6 rato 12. Neste protocolo de informação detalhada sobre as tensões para ser usado e o tamanho do eléctrodo de pá para uma maior especificidade é fornecido. Além disso, as informações sobre o ventrículo para ser preenchido, juntamente com as recomendações para a quantidade de solução de plasmídeo e a posição do eléctrodo é fornecido. A indicação das informações de posição detalhada em um mapa e de uma maior visualização destas posições permite específica simples e eficiente in utero eletroporação do córtex retrosplenial, o córtex motor, o córtex somatossensorial, o córtex piriforme t,cornu Ammonis ele 1-3, o giro denteado, o estriato, o núcleo septal lateral, no tálamo e hipotálamo.

<table><tbody><tr><td>EndoFree Plasmid Maxi Kit</td><td>QIAGEN</td><td>12362</td><td></td></tr><tr><td>Fast Green</td><td>Roth</td><td>0301.1</td><td></td></tr><tr><td>pCAGGS</td><td>Addgene</td><td></td><td></td></tr><tr><td>borosilicate glass capillaries (0.8-0.9 mm diameter)</td><td>Wold Precision Instrument Inc.</td><td>1B100F-4</td><td></td></tr><tr><td>Isoflurane (Forene)</td><td>Abbott</td><td>PZN 4831850</td><td></td></tr><tr><td>Carprofen (Rimadyl)</td><td>Pfizer GmbH</td><td>approval number: 400684.00.00</td><td></td></tr><tr><td>eye ointment (Bepanthen Augen und Nasensalbe)</td><td>Bayer&amp;nbsp;</td><td>PZN 01578681</td><td></td></tr><tr><td>0.9% benzyl alcohol 0.9% saline solution</td><td>Pharmacy&lt;br/&gt; of the University Medical Center Mainz</td><td></td><td></td></tr><tr><td>gauze (ES-Kompressen)</td><td>Hartmann</td><td>407835</td><td></td></tr><tr><td>sterile 5-0 Perma-Hand Silk Suture</td><td>Ethicon Johnson &amp;amp; Johnson</td><td>K890H</td><td></td></tr><tr><td>ring forceps 1/ 1.5 mm</td><td>Fine Science Tools</td><td>11101-09</td><td></td></tr><tr><td>ring forceps 4.8/ 6 mm</td><td>Fine Science Tools</td><td>11106-09</td><td></td></tr><tr><td>ring forceps 2.2/ 3 mm</td><td>Fine Science Tools</td><td>11103-09</td><td></td></tr><tr><td>Adson Forceps-Serrated Straight 12 cm</td><td>Fine Science Tools</td><td>1106-12</td><td></td></tr><tr><td>IrisScissors-Delicate Straight-Sharp/Blunt 10 cm</td><td>Fine Science Tools</td><td>14028-10</td><td></td></tr><tr><td>Mayo-Stille Scissors-Straight 15 cm</td><td>Fine Science Tools</td><td>14012-15</td><td></td></tr><tr><td>Dumont #5 Forceps-Inox</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Castroviejo NeedleHolder-with Lock-Tungsten Carbide 14 cm</td><td>Fine Science Tools</td><td>12565-14</td><td></td></tr><tr><td>Elektroporator CUY21 SC&amp;nbsp;</td><td>Nepa Gene Co.</td><td></td><td></td></tr><tr><td>FST 250 Hot Bead Sterilizer</td><td>Fine Science Tools</td><td>18000-45</td><td></td></tr><tr><td>Microgrinder EG-44</td><td>Narishige</td><td></td><td></td></tr><tr><td>P-97 Micropette Puller</td><td>Sutter Instrument Company</td><td>P-97</td><td></td></tr><tr><td>Platinum electrodes 650P 0.5 mm</td><td>Nepagene</td><td>CUY650P0.5</td><td></td></tr><tr><td>Platinum electrodes 650P 3 mm</td><td>Nepagene</td><td>CUY650P3</td><td></td></tr><tr><td>Platinum electrodes 650P 5 mm</td><td>Nepagene</td><td>CUY650P5</td><td></td></tr><tr><td>Platinum electrodes 650P 10 mm</td><td>Nepagene</td><td>CUY650P10</td><td></td></tr><tr><td>Anesthesia system</td><td>Rothacher-Medical GmbH</td><td>CV-30511-3 Vapor 19.3</td><td></td></tr><tr><td>Heating plate</td><td>Rothacher-Medical GmbH</td><td>HP-1M</td><td></td></tr><tr><td>Temperature Controller 220V AC</td><td>Rothacher-Medical GmbH</td><td>TCAT-2LV</td><td></td></tr></tbody></table>

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.

A Figura 2, mostra exemplos para a específica in utero electroporação das regiões em desenvolvimento para o córtex retroesplenial, o córtex motor, o córtex somatossensorial, o córtex piriforme, o cornu Ammonis 1-3, o giro denteado, o estriato, o núcleo septal lateral , o tálamo eo hipotálamo. Os resultados das transfecções são mostrados a seguir ao ângulo recomendado (Figura 2). Para melhor visualização dos ângulos in vivo a posição do eléctrodo (0...

Watch this Scientific Journal Video about Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse at JoVE.com

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 ...

cortex-hippocampus-thalamus-hypothalamus-lateral-septal-nucleus

Research

JoVE Journal

Neuroscience

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

27.9K visualizações.  University Medical Center, JGU Mainz. 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.

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

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

In Utero injeção intraventricular e eletroporação de embriões de camundongos E15

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

Biologia

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 ...

Neurociência

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 ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Expansão da embrionárias e adultas células-tronco neurais por<em&gt; In Utero</em&gt; Eletroporação ou injeção estereotáxica Viral

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

No útero e ex eletroporação in vivo para Expressão Gênica em células ganglionares da retina do rato

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

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 ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

Subtipo seletivo Eletroporação de Cortical Interneurônios

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

In Utero Eletroporação Abordagens para estudar a excitabilidade neuronal subpopulações e Conectividade uma única célula

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Double In Utero Electroporation to Target Temporally and Spatially Separated Cell Populations

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian corticogenesis. This procedure takes advantage of the brain ventricles to allow the introduction of DNA of interest and uses a pair of electrodes to direct the entrance of the genetic material into the cells lining the ventricle, the neural stem cells. This method allows researchers to label the desired cells and/or manipulate the expression of genes of interest in those cells. It has multiple applications, including assays targeting neuronal migration, lineage tracing, and axonal pathfinding. An important feature of this method is its temporal and regional control, allowing circumvention of potential problems related with embryonic lethality or the lack of specific CRE driver mice. Another relevant aspect of this technique is that it helps to considerably reduce the economic and temporal limitations that involve the generation of new mouse lines, which become particularly important in the study of interactions between cell types that originate in distant areas of the brain at different developmental ages. Here we describe a double electroporation strategy that enables targeting of cell populations that are spatially and temporally separated. With this approach we can label different subtypes of cells in different locations with selected fluorescent proteins to visualize them, and/or we can manipulate genes of interest expressed by these different cells at the appropriate times. This strategy enhances the potential of in utero electroporation and provides a powerful tool to study the behavior of temporally and spatially separated cell populations that migrate to establish close contacts, as well as long-range interactions through axonal projections, reducing temporal and economic costs.

Double in utero electroporation allows targeting cell populations that are spatially and temporally separated. This technique is useful to visualize interactions between those cell populations using fluorescent proteins in normal conditions but also after functional experiments to perturb genes of interest.

Dupla em Eletroporação utero para atingir populações celulares temporais e espacialmente separadas

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian ...

Murine In Utero Electroporation

In utero electroporation is an important technique for studying the molecular mechanisms that guide the proliferation, differentiation, migration, and maturation of cells during neural development. Electroporation enables the rapid and targeted delivery of material into cells by utilizing electrical pulses to create transient pores in cell membranes. Although electroporation has traditionally been used in in vitro studies, scientific advancements have now broadened its utilization to intact organs, such as those found in mouse embryos developing in utero. This video will introduce the key principles behind in utero electroporation in addition to reviewing the basic surgical techniques required to access developing embryos within a pregnant rodent. Details of the injection and electroporation steps are provided along with important considerations for directing gene delivery to specific brain regions. Finally, neurobiological applications of in utero electroporation are presented, such as investigating how specific genes contribute to neural development and how connections form between developing neurons.

Murine In Utero Electroporation

Eletroporação Murina In Utero

In utero electroporation is an important technique for studying the molecular mechanisms that guide the proliferation, differentiation, migration, and ...

Específicos do Striatum Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- e

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