Os autores gostariam de agradecer a Jane Johnson para o Math1, Ngn1 e anticorpos Ptf1a e Connie Cepko para o pCAG :: GFP plasmídeo. Este trabalho foi financiado pelo NIH R15 1R15HD059922-01.

1. Preparativos Antes da Eletroporação <ol><li> Amplificar o DNA de eletroporação por uma preparação maxi (Prime-It ou Qiagen). A concentração do ADN deve ser no mínimo de 1 mg / mL para a absorção eficiente. </li><li> Remover 495 uL de ADN e de mistura com 5 ul de 0,01% Verde Rápido em 1 X PBS (solução salina tamponada com fosfato) num tubo de microcentrífuga. </li></ol> 2. Colheita embrionário <ol><li> Estabelecer acasalamentos programados de CD-1 camundongos (Harlan). Verificar a presença de tampões vaginais e consideram a data um rolhão vaginal é observado como dia embrionário (E) 0,5. Os embriões serão colhidas 11 dias após visualização ficha (E11.5). </li><li> Coloque ferramentas esterilizados em etanol 70%. Tratar uma câmara de fluxo laminar com luz UV durante pelo menos uma hora antes do uso. Pulverização capô, bandeja de dissecação, e dissecando escopo com etanol 70%. Pré-aqueça 1 X PBS a 37 ° C. </li><li> Em E11.5 eutanásia a mulher de acordo com condições aprovadas pelo InstitutComitê ional para o Cuidado e Uso de Animais (ICCUA). Coloque bandeja de dissecação na capela de fluxo laminar. Pulverizar para baixo do abdômen da fêmea com etanol 70%. </li><li> Abrir a cavidade peritoneal e pino as paredes para a bandeja de dissecação. Retirar os cornos uterinos de modo que ele repousa no interior da cavidade peritoneal. </li><li> Corte com cuidado abrir a parede do corno uterino. Use uma colher de 20 mm (Belas Científicos) para remover suavemente o embrião no saco vitelino longe da placenta. Embriões lugar em um prato de cultura 100 milímetros tecido pré-preenchido com 10 mL de estéril 1 X PBS que foi pré-aquecido em 2,2. </li><li> Repita o procedimento para todos os embriões presentes. </li></ol> 3. Electroporação de Embriões E11.5 (Figura 1) <ol><li> Usando uma colher de 20 mm, transferência do embrião primeiro a um prato milímetros novo 100 pré-preenchido com 10 mL de 1XPBS estéreis que foi pré-aquecido a 37 ° C. </li><li> Use uma pinça de 11 cm com uma ponta de 0,05 mm x 0,02 (Belas Científicos) para cuidadosamente remover e descartar o saco vitelino. </li><li> Embrião posição de modo que seu lado dorsal está virada para cima para que ele lembra o desenho animado na Figura 1A. Use pás 7 mm de eletrodos (Harvard Apparatus) suavemente para segurar o embrião. Os eléctrodos deverão ser posicionados em ambos os lados do tubo neural ao nível do ventrículo rombencéfalo quarto. O ventrículo é visível a olho nu, mas utilizando um microscópio de dissecção pode facilitar a colocação de pás precisas. Posicionamento das pás é crítico para determinar a região que recebe o DNA electroporado. Se o lábio inferior rômbica (MID), da parte posterior do cérebro embrionário dorsal é desejada, pás deve ser o local de tal forma que eles estão directamente flanqueando a parte mais larga da abertura quarto ventrículo. </li><li> Use uma seringa cc 1 a elaborar o DNA plasmídeo misturado com 0,01% de verde rápido. 500 uL da mistura deve ser suficiente para a electroporação de pelo menos 8-10 embriões (ver 3.5). </li><li> Gentilmente perfurar o ov roofplateerlying quarto ventrículo com uma agulha de 25G tuberculina 5/8 ligada à seringa 1 cc e injectar a mistura de ADN-corante para o ventrículo. Injecções bem sucedidos são caracterizados pela mistura de ADN de corante de enchimento do sistema ventricular inteiro (Figura 1C). A quantidade de DNA de corante-mistura tipicamente injectado é inferior a 50 uL. As quantidades exactas são variáveis ​​entre embriões como uma porção da mistura tende a escapar para fora do ventrículo para o PBS em torno do embrião. Um meio alternativo de fornecer a mistura de corante-DNA seria por meio de acessar o sistema ventricular por punção do véu que cobre o mesencéfalo. Novamente, as injecções bem sucedidos são caracterizados pela mistura de ADN de corante de enchimento do sistema ventricular inteiro. </li><li> Entregar cinco pulsos quadrados com um gerador de pulso elétrico (BTX) e 7 pás de eletrodos mm. Cada pulso é de 50 V duração de 5 ms por pulso com 500 ms entre cada pulso. O tecido mais próximo do eléctrodo carregado positivamente irá então Take se o plasmídeo. </li></ol> 4. Cultura de Embriões <ol><li> Em uma câmara de fluxo laminar, encher os poços exteriores de um prato de cultura de 12 poços com 2 mL de DMEM/F12 meios suplementados com 10% de soro fetal de bovino, 5% de soro equino, 1% de glutamina, 1% de penicilina / estreptomicina que foi pré-aquecido a 37 ° C. As condições de cultivo foram adaptados a partir de Diego e colegas 15. </li><li> Em embriões de escoamento laminar de aperto da capa de midsection (abaixo do coração) com uma pinça e remover porção posterior do embrião. Coloque a porção anterior em um dos poços cheios do prato de cultura 12 poços. </li><li> Repita o procedimento para todos os embriões. Encha as cavidades apenas exterior da placa de 12 poços para evitar a contaminação das culturas. </li><li> Embriões cultura em um 37 ° C incubadora com 5% de CO 2. Expressão do plasmídeo electroporado deve ser observável no prazo de 24 horas. </li><li> Caso mais vezes cultura ser desejado, encher os poços para fora de uma placa de 12 poço novo com 2 mL deo material usado na versão 4.1. Com uma colher esterilizada transferir os embriões para um poço na placa de novo. Coloque de volta para a 37 ° C incubadora. Cultura para até 48 hr é possível. </li><li> Quando o tempo de cultura desejada é alcançada, fixar os embriões para análise (ver abaixo). </li></ol> 5. Preparação de Embriões de Análise <ol><li> Lavar embriões em 1 X PBS a 4 ° C durante cinco minutos. Repita. </li><li> Corrigir embriões para análise em paraformaldeído a 2% (PFA) em 1XPBS durante 2 horas a 4 ° C. </li><li> Lavar embriões em 1 X PBS a 4 ° C durante cinco minutos. Repita. </li><li> Equilibre embriões em sacarose a 30% em 1 X PBS durante a noite a 4 ° C. </li><li> Incorporar embriões em Optimal composto de corte de temperatura (OCT) usando um banho de gelo / etanol seco. Os embriões podem ser armazenados a -20 ° C. </li><li> Embriões seção em um criostato (Leica) em 30 seções mM e montar em lâminas (VWR, Superfrost Plus). Armazenar a -20 ° C. </li></ol> 6. ImmuAnálise imunohistoquímica <ol><li> A imuno-histoquímica foi realizada como descrito em 16. Diluições de anticorpos primários utilizados para este estudo incluem coelho α-GFP (Invitrogen) 1:2500; rato α-Mash1 (BD Biosciences) 1:100; coelho α-Ngn1 (Jane Johnson) 1:5000; coelho α-Ptf1a (Jane Johnson ) 1:2500;-coelho Math1 (Jane Johnson) 1:100. Incubar corrediça lado espécime plana, em cima de uma bandeja de coloração a 4 ° C durante a noite. </li><li> As lâminas foram analisadas num microscópio composto (Olympus BX51). </li></ol> 7. Os resultados representativos Um diagrama esquemático na Figura 1A mostra a experiência electroporação. A Figura 1B mostra uma vista sagital de um embrião E11.5 antes da manipulação. O embrião mesmo após a injecção do CAG plasmídeo contém :: GFP em 0,01% Verde Rápido é mostrado na Figura 1C e um embrião representante unfixed exibindo GFP unilateralexpressão no cérebro posterior dorsal cultura hr 24 seguinte é mostrado na Figura 1D. A extensão da área do LRL que com êxito é electroporado é variável e parece ser altamente dependente de posicionamento dos eléctrodos. Nos nossos estudos, verificou-se que 52 de 65 (80%) dos embriões eletroporados expressa com sucesso GFP. O tecido foi considerada com sucesso electroporado se era positivo para GFP em regiões localizadas sobre várias secções sequência de fixação e análise imuno-histoquímica (ver abaixo). Os embriões que não cumpriram este critério foram classificados como tentativas fracassadas de eletroporação. Além disso avaliação da eficiência de electroporação pode ser determinado através da realização de imuno-histoquímica contra GFP em secções transversais de embriões eletroporados ao nível do quarto ventrículo. A Figura 2 mostra representativos secções seriadas de um embrião exibindo unilateralExpressão da GFP 2A. Figura mostra um diagrama esquemático que ilustra idealizada que o lado esquerdo do embrião era para o eléctrodo positivo. Secções transversais (em níveis representados por meio do desenho da Figura 2A) revelam expressão da GFP localizada exclusivamente no lado esquerdo do tecido rombencéfalo (Figuras 2C e 2D). A área electroporado do LRL não alargar o eixo anterior-posterior inteira do tubo neural como exame das secções 300 mm ou maior rostral à mostrada na Figura 2C não expressa a GFP (Figura 2B). A utilidade deste ensaio para a manipulação da expressão do gene é dependente da estabilidade dos domínios de expressão para as proteínas endógenas. O LRL tem sido caracterizado como possuindo domínios progenitoras únicas caracterizadas pela expressão diferencial de factores de transcrição proneural (revisto em 1).Um subconjunto destes factores (Mash1, Math1, Ngn1, e Ptf1a) foram escolhidos para análise devido à sua proposta e / ou funções caracterizadas na especificação de precerebellar subtipos neurais no LRL, um objecto de estudos futuros 16-18. Todos os quatro proteínas têm domínios de expressão altamente característicos no cérebro posterior caudal em E11.5 16-18. Observou-se que os embriões que foram colocados culturas não conseguiu aumentar em dimensão e também falhou para iniciar a produção do epitélio do plexo coróide e invaginação da LRL e roofplate, eventos que ocorrem entre morfológicas E11.5 e E12.5 5. Com base nestas observações, foi determinado que o desenvolvimento normal em estes embriões foi interrompido ou grosseiramente retardada e controlo comparável para os embriões cultivados deve ser embriões não cultivadas em E11.5. Para garantir que a cultura ea eletroporação não perturbar os níveis de proteínas endógenas, foram analisados ​​quatro proteínas diferentes por immunohistochemistry (IHC) em 34 embriões diferentes que foram electroporadas e em seguida cultivadas durante pelo menos 24 horas. Tabela I mostra o número de embriões analisados ​​para cada marcador ea percentagem dos embriões analisados ​​que retinham os níveis de proteína normal. A Figura 3 mostra representativa IHC dados a partir de duas das proteínas analisadas, Mash1 (Figuras 3A e 3B) e Math1 (Figuras 3C e 3D). Observou-se que a maioria dos embriões retida níveis normais de electroporação seguinte expressão e cultura (Figuras 3B e 3D), em comparação com os embriões de controlo nas E11.5 (Figuras 3A e 3C). Importante, os domínios de expressão característicos destas proteínas não foram perturbado. <img src="/files/ftp_upload/3983/3983fig1.jpg" alt="A Figura 1"/> Figura 1. Electroporação de Embriões em E11.5. Esquema (A) de thexperiência e eletroporação. Um embrião E11.5 é isolado e do plasmídeo de expressão em 0,01% Verde Rápido é injectado no quarto ventrículo. O embrião é, então, flanqueado por pás de eléctrodos e submetido a um pulso de 50 V, antes de serem colocadas em cultura (B) vista sagital de um embrião E11.5 antes da injecção. (C) O embrião E11.5 mesmo após a injecção do plasmídeo em 0,01% Verde Rápido. (D) expressão rombencéfalo unilateral de GFP observada em embriões E11.5 após 24 horas de cultura. mb mesencéfalo; 4v quarto ventrículo. <img src="/files/ftp_upload/3983/3983fig2.jpg" alt="A Figura 2"/> Figura 2. Expressão de GFP em Tecido eletroporados. (A) O desenho à esquerda mostra a colocação dos eletrodos em torno de um embrião E11.5. Dos desenhos mostra a absorção do meio e da expressão da GFP plasmídeo no lado esquerdo do embrião. Desenhos animados em direito é esquemático de uma seção transversal idealizada tomadas através do embrião no levels denotada por linhas pretas em meio caricatura. (B-D) Imunohistoquímica para GFP em seções transversais através de um embrião E11.5 eletroporados após 24 horas de cultura. As setas indicam o roofplate (RP) que retém o anticorpo secundário. As imagens são tomadas com uma ampliação de 10X. Os níveis relativos das secções mostradas são representadas pelas linhas horizontais através do desenho do meio em (A). mb mesencéfalo; ventrículo l 4v quarto; rp-roofplate. <img src="/files/ftp_upload/3983/3983fig3.jpg" alt="A Figura 3"/> Figura 3. Expressão de proteínas endógenas no lábio inferior rômbico A imuno-histoquímica para Mash1 (A e B) ou Math1 (C e D), comparando secções transversais de embriões E11.5 que não foram cultivadas (A, C) com embriões que foram electroporadas com CAG.: : GFP e cultivadas durante 24 h (B e D). Imagens tomadas com uma ampliação de 10X. <table border="1"><tbody><tr><td> Transcripti Proneuralem fatores analisados </td><td> Número de embriões analisados </td><td> Percentagem de retenção Padrões expressão normal </td></tr><tr><td> Math1 </td><td> 15 </td><td> 86,7% </td></tr><tr><td> Mash1 </td><td> 12 </td><td> 83,3% </td></tr><tr><td> Ngn1 </td><td> 7 </td><td> 71,4% </td></tr><tr><td> Ptf1a </td><td> 6 </td><td> 100% </td></tr></tbody></table> Percentagem Tabela I. de Embriões eletroporados e Cultivadas retenção normais Domínios transcrição Proneural fator na LRL.

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A técnica in vitro electroporação apresentado neste estudo é uma nova metodologia que pode ser utilizada de forma eficiente para manipular a expressão do gene em embriões de menos de 12 dias de gestação. Colocação dos embriões em cultura permite a expressão do gene introduzido e contorna a letalidade observada quando embriões eletroporados for permitido permanecer in vivo. Esta técnica permite a manipulação da expressão gênica em células progenitoras embrionárias que anteri...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> Criostato </td><td> Leica </td><td> CM-1850 </td><td></td></tr><tr><td> Biologie ponta Dumoxel tratados DUMONT fórceps </td><td> Belas Científicos </td><td> 11252-30 </td><td></td></tr><tr><td> 20 milímetros colher perfurada MORIA </td><td> Belas Científicos </td><td> 10370-17 </td><td></td></tr><tr><td> ECM 830 Praça Gerador de Eletroporação Onda </td><td> BTX (VWR) </td><td> 47745-928 </td><td></td></tr><tr><td> Harvard Apparatus 7 Tweezertrodes mm * Eletrodos </td><td> BTX (Fisher) </td><td> BTX450165 </td><td></td></tr><tr><td> Fisher Isotemp incubadora de CO 2 </td><td> Pescador </td><td> 1325525 </td><td></td></tr><tr><td> NAPCO CO Regulador de Gás 2 </td><td> Pescador </td><td> 15497020 </td><td></td></tr><tr><td> 12 placas de cultura de tecidos </td><td> BD Falcon (Fisher) </td><td> 877229 </td><td></td></tr><tr><td> HyClone líquido Meios DMEM/F-12 (1:1); com L-glutamina e HEPES; 500mL </td><td> Thermo Scientific (Fisher) </td><td> SH3002301 </td><td></td></tr><tr><td> HyClone * Doador soro eqüino </td><td> Thermo Scientific (Fisher) </td><td> SH3007402 </td><td></td></tr><tr><td> Soro fetal bovino, Calor, qualificado inativada </td><td> Invitrogen </td><td> 16140-063 </td><td></td></tr><tr><td> cellgro * 10.000 UI de penicilina, estreptomicina 10.000 g / mL </td><td> Mediatech (Fisher) </td><td> MT-30-002-CI </td><td></td></tr><tr><td> HyClone * L-Glutamina L-Glutamina, 200mM em NaCl 0,85% </td><td> Thermo Scientific (Fisher) </td><td> SH3003401 </td><td></td></tr><tr><td> Fast-Verde </td><td> Pescador </td><td> AC41053-0250 </td><td> 0,01% </td></tr></tbody></table>

in vitro electroporation of the lower rhombic lip of midgestation mouse embryos

O lábio rômbico é um neuroepitélio embrionário localizado no cérebro posterior na junção entre o tubo neural eo roofplate do quarto ventrículo (revisto em 1). O lábio rômbico pode ser subdividido no lábio superior rômbica (URL), que engloba rhombomere 1 (R1) e gera os neurónios do cerebelo eo lábio inferior rômbica (MID), que dá origem a diversas linhagens de tronco cerebral neuronais 2-4. Derivados LRL incluem os neurónios auditivos dos núcleos coclear e aqueles dos núcleos precerebellar que estão envolvidos na regulação do motor e do equilíbrio de controlo 5-8. Neurogênese a partir do LRL ocorre ao longo de uma grande janela temporal que engloba dias embrionários (E) 9,5-16,5 5, 9. Diferentes linhagens neuronais emergir do LRL como células pós-mitóticas (ou nascem) durante diferentes dias de desenvolvimento durante esta janela neurogênica. Electroporação de construções de expressão de genes pode ser usado paramanipular a expressão do gene em células progenitoras LRL e potencialmente pode mudar o destino dos neurônios produzidos a partir desta região 10-12. Alterando a expressão gênica de células progenitoras LRL no rato através de eletroporação in utero tem sido muito bem sucedida para a manipulação de linhagens nascidos no dia embrionário E12.5 ou mais tarde, 10, 12-14. Em electroporações útero antes E12.5 não tiveram sucesso devido principalmente à letalidade associada a punção da roofplate quarto ventrículo, um passo necessário na entrega de DNA exógeno que é electroporados para o MID. No entanto, muitos LRL linhagens derivadas surgem a partir do LRL mais cedo do que E12.5 9. Estas linhagens anteriormente nascidos incluem os neurónios que compõem a reticular lateral, cuneate externo, e os núcleos olivar inferior do sistema de precerebellar que funcionam de modo a ligar as entradas a partir da medula espinal e do córtex para o cerebelo 5. A fim de manipular expressão na LRLde embriões com menos de E12.5, desenvolvemos um sistema in vitro em que os embriões são colocados em cultura na sequência de electroporação. Este estudo apresenta um método eficiente e eficaz para manipular a expressão de genes dos progenitores LRL em E11.5. Embriões electroporado com a proteína verde fluorescente (GFP) accionada a partir do promotor CAG amplamente activa reprodutivelmente expressa GFP após 24 horas de cultura. Um aspecto crítico da presente ensaio é que a expressão do gene só podem ser alterados por causa da expressão do gene exógeno e não por causa de efeitos secundários que resultam da electroporação e técnicas de cultura. Foi determinado que os padrões de expressão de genes endógenos permanecer intacta em embriões eletroporados e culta. Este ensaio pode ser utilizado para alterar o destino das células que emergem do LRL de embriões com menos de E12.5 através da introdução de plasmídeos para a superexpressão ou derrubar (através RNAi) de diferentes pró-neurais fatores de transcrição.

Watch this Scientific Journal Video about In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos at JoVE.com

In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

Este estudo descreve o desenvolvimento de um<em&gt; In vitro</em&gt; Técnica de electroporação que permite a manipulação da expressão do gene no lábio inferior rômbico de embriões midgestation.

Em Eletroporação vitro do lábio inferior rômbico de embriões de camundongos Midgestation

The rhombic lip is an embryonic neuroepithelium located in the hindbrain at the junction between the neural tube and the roofplate of the fourth ventricle ...

in-vitro-electroporation-lower-rhombic-lip-midgestation-mouse

Research

JoVE Journal

Neuroscience

In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

10.3K visualizações.  University of Illinois at Springfield. Este estudo descreve o desenvolvimento de um In vitro Técnica de electroporação que permite a manipulação da expressão do gene no lábio inferior rômbico de embriões midgestation.

Vídeo: In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

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 ...

Neurociência

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

Quantificar a atividade de Cis Elements-Regulação na retina do rato por Eletroporação Remoção

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

Em Eletroporação vivo de Retina do rato em desenvolvimento

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

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 ...

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 ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

Eletroporação do rombencéfalo rastrear Trajetórias axonal e Metas Synaptic no embrião de galinha

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

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 ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

Um<em&gt; In-vitro</em&gt; Preparação de neurônios entéricos isolados e células gliais do plexo mioentérico do Rato Adulto

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.