Agradecemos Imprensa Humana permissão para publicar a microinjeção figura fabricação pipeta que apareceu originalmente na página 130 da referência 11; Larry Dlugas e Steven Wong, OHSU Departamento de Comunicações Educacionais, para videografia; Larry Dlugas para vídeo design e edição; Adam M. O &#39;Quinn, Designer Sênior, Trion / Envirco para projetar nossa capela de fluxo laminar horizontal personalizado e Les Goldsmith para fornecer o esquema técnico; Victor Monterroso, MV, MS, PhD e Tom Chatkupt, DVM, OHSU Departamento de Medicina Comparada, para orientação com a nossa animais protocolo de atendimento, técnicas cirúrgicas e analgesia profilática regime; Marcel Perret-Gentil, DVM, MS, para partilhar a sua apostila sobre técnicas de sutura veterinários; Edward Porsov, MS, para projetar nossa Adobe Premiere Pro vídeo estação de trabalho do computador microscopia, e Leah e Branco Jonas Hinckley de LNS Caption (Portland, OR). Este trabalho foi financiado por doações do Instituto Nacional de Surdez e outrDistúrbios r Comunicação: DC R01 008.595 008.595 R01 e DC-04S2 (JB) e P30 DC005983 (Audição do Oregon Research Center Núcleo Grant, Peter Gillespie, Investigador Principal).

1. A laparotomia ventral <ol><li> Anestesiar uma barragem cuja embriões são no dia embrionário 11,5 (E11.5; meio-dia no dia um rolhão vaginal é detectado é dia de desenvolvimento embrionário 0,5), por injecção intraperitoneal de pentobarbital de sódio da solução anestésica (7,5 uL por peso de corpo grama). Trabalhando solução anestésica: 180 uL de 50 mg / mL solução de sódio pentobarbital; 100 uL de etanol absoluto; 320 uL de 65 mg / mL de sulfato de magnésio aquoso (modula tom uterina); e 400 uL de propileno glicol (miscível veículo com uma solução aquosa e orgânica componentes). </li><li> Avaliar a completude da anestesia através da realização de testes de estímulos nocivos: squeeze pata; pitada cauda; e piscar de resposta ao toque bochecha e vibrissas. Aplicar pomada oftálmica estéril para as córneas. </li><li> Raspar o pêlo da área abdominal suprapúbica para a caixa torácica com uma tesoura e uma lâmina fina (Oster lâmina # 40). Desinfectar o abdómen com etanol a 70%, iodo povidona 10% (Betadine), umnd etanol 70%, seqüencialmente. Lugar do mouse abdômen virado para baixo em um pano estéril e em seguida, definir sobre uma almofada de aquecimento ou chapa quente (37 ° C) por 2-5 minutos. </li><li> Faça uma incisão na pele abdominal na linha média ventral com bola com ponta de tesoura. Alargue a incisão para 10-14 mm. Identificar a linha alba, um avascular, faixa de tecido conjuntivo branco ao longo da linha média ventral da parede abdominal. Faça uma incisão na linha alba com a bola derrubou uma tesoura e estender a incisão 10-14 mm. Imediatamente irrigar o abdômen com pré-aquecido (37 ° C) solução de Ringer com lactato. </li><li> Exteriorizar as duas pontas do útero com uma pinça de anel, sem aplicar pressão excessiva sobre os locais de implantação. Irrigar os cornos uterinos externalizados com pré-aquecido, solução de Ringer com lactato. </li></ol> 2. Microinjeção transuterina <ol><li> Fabricar uma espessura de parede, vidro borosilicato pipeta de microinjeção capilar com as seguintes características: 12-16 μm de diâmetro exterior e um bisel de 20 graus. Em um extrator da pipeta Sutter P-97 com filamento pequena caixa, use o seguinte programa: calor = teste de rampa mais 3 unidades; pressão = 200; puxar = 0; velocidade = 46; tempo = 100). Com o beveler Sutter BV-10, utilizar o disco (ouro) 104C abrasivo para pipetas de grande diâmetro. Ressuspender o plasmídeo de expressão em ug 3-4 / uL em fosfato de cálcio livre tamponado salino (pH 7,2-7,4). Adicionar verde rápido cristalina ao concentrado DNA vórtice, suavemente durante 30 segundos, e girar a 10.000 g por 10 segundos. A quantidade mínima de verde rápido necessário para controlar visualmente a eficácia da injecção é determinada empiricamente. Aterrar a pipeta biselada com o DNA / solução de verde rápido. Ligue o pipeta microinjecção carregado para o titular da pipeta da pressão do injector (Picospritzer usando&gt; azoto 99% puro como fonte de gás). </li><li> Transilluminate do útero com baixa intensidade, a luz de halogéneo para visualizar o embrião no seu local de implantação. Identificar o beating coração, vesículas cerebrais, brotos, ventrículo ª nascente 4 do rombencéfalo e oculares. Irrigar o útero a cada 2 minutos, com pré-aquecido e solução de lactato de Ringer para manter a hidratação. Aplique uma leve pressão sobre o útero para reorientar o embrião e identificar os pontos anatômicos mencionados acima. </li><li> Oriente o embrião para identificar a veia principal, cuja cabeça anterior e posterior do flanco ramos do território mesenquimal em que o otocyst reside. O otocyst adequada não pode ser visto por transiluminação do útero. O otocyst está localizado a meio caminho entre os ramos anterior e posterior da veia cabeça primário que, juntamente com o tronco principal da veia, formar a forma dos pilares ou postes de um campo de futebol americano. O otocyst está a meio caminho entre os postes. </li><li> Inserir a pipeta de injecção através do útero de uma trajectória em linha com a localização presuntiva do otocyst. Pulsar o microinjector uma vez, depois de passar através do uternos visualizar o corante marcador, a localização aproximada da ponta da pipeta. Avançar a pipeta sob controle micrômetro e pulse novamente para avaliar a profundidade. Avançar a pipeta no mesênquima lateral da cabeça e pulso repetidamente. Segmentação otocyst sucesso irá revelar a forma afilada do ducto endolinfático dorsalmente ea forma de concha de vieira do vestíbulo. Solte a pressão sobre o útero, remover a pipeta do embrião / útero em um movimento, e imediatamente irrigar o útero com pré-aquecido, solução de Ringer lactato. </li></ol> 3. Em electroporação in vivo <ol><li> Irrigar o útero com solução de Ringer com lactato. Solução recentemente aplicado de Ringer com lactato é necessária para os eletrodos eletricamente par de remo de estilo para o útero. Umedeça as superfícies dos eletrodos de tungstênio com Ringer com Lactato. Centro da otocyst injetado no caminho dos eletrodos. Gentilmente comprimir o útero com o pa eletroporaçãoddles. O cátodo está em contacto com a parede uterina lateral à otocyst injectado eo ânodo está em contacto com a parede uterina adjacente ao otocyst não injectada. Dispara um trem de pulso de onda quadrada com o interruptor de pedal na electroporator. Eletroporação parâmetros são: 5, 50 ms pulsos em 43 volts por impulso e 950 ms de atraso interpulso. Imediatamente irrigar o útero após o trem de pulsos é entregue. Gravar a corrente fornecida ao tecido: 60-100 mAmps é suficiente para transfectar óticos progenitores epiteliais. Injectar e electroporate 4-6 embriões, E11.5 por barragem. </li><li> Executar um segundo, microinjecção independente transuterina de fluorescente aquosa de dextrano (Alexa Fluor 488 se o plasmídeo de expressão codifica uma proteína vermelho fluorescente ou Alex Fluor 594 se o plasmídeo de expressão codifica uma proteína fluorescente verde) para o ventrículo th 4 desses embriões cujo interior orelhas são injetado com precisão e pelo menos 60 mAmps de corrente por pulso era delivered durante eletroporação. O dextran fluorescente será detectado ao nascer no cérebro posterior e permitir a seleção de filhotes cujo interior ouvidos foram manipulados durante a embriogênese (ver 3.5). </li><li> Irrigar os cornos uterinos com Ringer com Lactato. Reinserir os cornos uterinos para dentro da cavidade abdominal. Lave a cavidade uterina com 2-4 mL de pré-aquecido, solução de Ringer com lactato e permitir que o excesso seja drenada do local da incisão para a cortina estéril. Substitua o draping com material seco e estéril. Suturar a parede abdominal com uma agulha não cortante e uma sutura reabsorvível 6-0. Nós preferimos um ponto corrido, travando cada ponto outro, tanto para a parede abdominal e da pele. </li><li> Seque pele das barragens e administrar um não-esteróides anti-inflamatórias, tais como Meloxicam por injecção subcutânea. Volte a represa para a gaiola de recuperação pré-aquecido em uma cortina estéril. Monitore e registre as respirações das barragens, a patência local da incisão e vagina de secreção sanguinolenta. O sangramento é rare, mas se presente e, sem trégua, a eutanásia da barragem, enquanto ela ainda está sob anestesia. Observe o tempo, ela recobra a consciência e as tentativas de deambular. Retorne a barragem para a colônia do mouse quando ela mostra sinais de comer e beber e começou a construir ninho. Tipicamente, isto ocorre no prazo de 12 horas. </li><li> No momento do nascimento (dia pós-natal 0), flash região rombencéfalo de cada filhote da ninhada para detectar o dextran fluorescente usando um microscópio de dissecação stereofluorescence com GFP ou Red Texas filtro definido como apropriado. Voltar para a barragem de lactação somente os filhotes que mostrar rotulagem rombencéfalo. </li></ol> 4. Os resultados representativos <img src="/files/ftp_upload/3653/3653fig1.jpg" alt="A Figura 1" /> Figura 1. Electroporação transferência de genes mediada para a cóclea em desenvolvimento. O otocyst E11.5 foi injectado com um plasmídeo de expressão de proteína de codificação verde melhorada fluorescente (EGFP) e electroporado (pulso tranos parâmetros: cinco, 43 pulsos volts a 50 ms / pulso e 950 ms de atraso interpulso). A) orelha Um representante interior a partir de um dia pós-natal 6 (P6) pup cuja otocyst foi injectado e electroporado em E11.5 demonstrando a expressão EGFP a partir da base através da espira média da cóclea. A parede lateral da cóclea foi removido da espira média e ápice só. Progenitores E11.5 que dão origem ao ápice não foram transfectadas. B) de montagem Whole imunocoloração da cóclea em (A) com o marcador de células ciliadas, miosina 7a (MYO7A), indica que a expressão EGFP segue a trajectória do órgão de Corti e é grosseiramente localizada ao cabelo epitélio célula-rolamento sensorial. C) Um representante da orelha interna de um embrião E18.5 cuja otocyst foi injetado e electroporados em E11.5. A projeção a laser confocal demonstra a expressão EGFP em MYO7A-positivos células sensoriais ciliadas. D) A projeção a laser confocal do epitélio coclear sensoriais do órgão de Corti E18.5 indicando EGFP expression nas células ciliadas internas (CCI), as células ciliadas externas (CCE), células internas phalangial (IPC), células pilar (PC), e células Deiters &quot;(cc). A barra de escala em (B) aplica-se a (A).

<ol>
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Transferência de genes para a orelha do rato em desenvolvimento interior: A orelha do rato interior se desenvolve a partir da placode ótica durante a primeira semana de pós-implantação desenvolvimento 12,13. Por dia embrionário 9,5 (E9.5), o placode tem invaginado e se transformou em uma vesícula cheia de líquido chamado otocyst 2. Precursores óticos na vesícula dar origem às células sensoriais e não-sensorial dentro do ouvido interno madura, bem como os neurónios que...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Caso esterilização Micro </td><td> Roboz </td><td> RS-9900a </td><td> 8X8.5X1.25 polegadas </td></tr><tr><td> Bola de ponta tesoura </td><td> Belas Ferramentas Ciência </td><td> 14109-09 </td><td></td></tr><tr><td> Fórceps do anel </td><td> Belas Ferramentas Ciência </td><td> 11106-09 </td><td> 4,8 milímetros ID/6mm OD </td></tr><tr><td> Tecido Pinça Adson </td><td> Belas Ferramentas Ciência </td><td> 11027-12 </td><td></td></tr><tr><td> Motorista agulha </td><td> Belas Ferramentas Ciência </td><td> 12502-12 </td><td></td></tr><tr><td> Bandeja Seringa Alergia </td><td> Becton Dickison </td><td> 305536 </td><td></td></tr><tr><td> Sutura 6-0 </td><td> Syneture </td><td> GL-889 </td><td> 0,7 sutura métrica gastrointestinal </td></tr><tr><td> Injeção de Ringer com lactato USP </td><td> Baxter </td><td> 2B2323 </td><td></td></tr><tr><td> Verde rápido </td><td> Sigma Aldrich </td><td> F7258 </td><td></td></tr><tr><td> Borosilicato capilar de vidro </td><td> Harvard Apparatus </td><td> 30-0053 </td><td></td></tr><tr><td> Solução de sódio Nembutal </td><td> OVATION Pharmaceuticals Inc. </td><td> NDC 67386-501-52 </td><td></td></tr><tr><td> MgSO4.7H2O </td><td> Fisher Scientific </td><td> M63-500 </td><td></td></tr><tr><td> Propilenoglicol </td><td> Fisher Scientific </td><td> P355-1 </td><td></td></tr><tr><td> Etanol </td><td> Sigma Aldrich </td><td> E7023-500 </td><td></td></tr><tr><td> Meloxicam </td><td> Boehringer Ingeheim </td><td> NADA 141-219 </td><td></td></tr><tr><td> Micropipeta Puller </td><td> Sutter Instruments </td><td> P-97 </td><td> FB255B filamento caixa; consultar Cookbook de pipeta de instrumentos de Sutter </td></tr><tr><td> Beveler microeletrodo </td><td> Sutter Instruments </td><td> BV-10 </td><td> Disco chanfradura 104C para pipetas grandes, consultar o manual do proprietário para biselamento teoria </td></tr><tr><td> Titular Micropipeta </td><td> Warner Instruments </td><td> MP-S15T </td><td> Para pipeta de 1,5 milímetros de diâmetro exterior e porta de pressão fêmea para tubagem Picospritzer. </td></tr><tr><td> Pinças estilo de eletrodo </td><td> Protech International Inc. </td><td> CUY650P5 </td><td> 5 mm de diâmetro exterior </td></tr><tr><td> Onda Quadrada Electroporator </td><td> Protech International Inc. </td><td> CUY21EDIT </td><td> Footpedal recomendada </td></tr><tr><td> PICOSPRITZER III </td><td> Parker Hannifin </td><td> 051-0500-900 </td><td> Footpedal recomendada </td></tr><tr><td> Micromanipulador Controle Manual </td><td> Harvard Apparatus </td><td> 640056 </td><td></td></tr><tr><td> Bancada de fluxo laminar horizontal limpa </td><td> Envirco </td><td></td><td> Modificações personalizadas para 630-10554 LF. Ver informações complementares para esquemática capô. </td></tr><tr><td> Leica stereofluorescence dissecando microcope com Lumencor motor de luz SOLA </td><td> Bartels e Stout e Lumencor </td><td> MZ10F com Lumencor motor de luz SOLA </td><td> Footpedals de enfocar o MZ10F e para acionar o motor de luz SOLA são recomendados </td></tr><tr><td> Alexa Fluor 594 Dextran </td><td> Invitrogen </td><td> D22913 </td><td> Aquoso, 10mg/ml </td></tr><tr><td> Alexa Fluor 488 Dextran </td><td> Invitrogen </td><td> D22910 </td><td> Aquoso, 10mg/ml </td></tr><tr><td> Enviro-dri </td><td> Pastor Pap Especialidadeers </td><td></td><td> <a href="http://www.ssponline.com" target="_blank">www.ssponline.com</a> </td></tr></tbody></table>

gene transfer to the developing mouse inner ear by in vivo electroporation

A orelha de mamífero interior tem 6 epitélios distinta sensorial: 3 cristas na ampolas dos canais semicirculares; máculas no utrículo e sáculo e do órgão de Corti na cóclea enrolada. O cristas e máculas conter células ciliadas vestibulares que transdução estímulos mecânicos para servir à especial sentido de equilíbrio, enquanto que as células ciliadas auditivas no órgão de Corti são os transdutores primários para ouvir 1. Especificação célula destino nestas epitélio sensorial e morfogênese dos canais semicirculares e cóclea ter lugar durante a segunda semana de gestação em camundongos e são praticamente concluída antes do nascimento 2,3. Estudos sobre o desenvolvimento da orelha interna do rato são rotineiramente realizados pela colheita de embriões transgênicos em diferentes estádios de desenvolvimento embrionário ou pós-natal para obter insights sobre a base molecular do celular e / ou morfológica fenótipos 4,5. Nós supomos que a transferência de genes para a orelha interna do rato em desenvolvimento no útero &lt;/ Em&gt; no contexto de ganho e perda de função de estudos representa uma abordagem complementar ao transgênese mouse tradicional para o interrogatório dos mecanismos genéticos subjacentes ao desenvolvimento do ouvido interno de mamíferos 6. O paradigma experimental para realizar estudos de expressão ectópica de genes na orelha do mouse desenvolvimento interior demonstrado aqui resolve-se em três etapas gerais: 1) laparotomia ventral; 2) microinjeção transuterina e 3) em eletroporação in vivo. Laparotomia ventral é uma técnica de sobrevivência do mouse cirúrgica que permite a exteriorização do útero para ter acesso experimental para os embriões implantados 7. Microinjecção transuterina é o uso de chanfradas micropipetas, capilares de vidro para introduzir plasmídeo de expressão para dentro do lúmen da vesícula ótica ou otocyst. In vivo electroporação é a aplicação de onda quadrada, directos impulsos de corrente para dirigir a expressão de plasmídeo em 8-10 células progenitoras. 11. Rato experimentais técnicas embriológicas pode ser difícil de aprender com prosa e imagens por si só. No presente trabalho, demonstramos os 3 passos no processo de transferência de genes. Mais criticamente, com a implantação de microscopia de vídeo digital para mostrar precisamente como: 1) identificar a orientação do embrião no útero; 2) reorientar embriões para alvejar as injeções para o otocyst; 3) microinject DNA misturado com calda de corante no otocyst nos dias embrionárias 11,5 e 12,5; 4) electroporate o otocyst injetada e 5) embriões rótulo eletroporados para a seleção pós-natal ao nascimento. Nós fornecemos exemplos representativos de sucesso transfectadas ouvidos internos, um guia ilustrado para as causas mais comuns de mistargeting otocyst; discutir como evitar erros comuns metodológicas e diretrizes presentes para escrever um in utero geno protocolo de transferência de cuidados com animais.

Watch this Scientific Journal Video about Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation at JoVE.com

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

A orelha interna do rato é um órgão sensorial placode derivado cujo programa é elaborado de desenvolvimento durante a gestação. Nós definimos uma<em&gt; In utero</em&gt; Técnica de transferência de gene consiste de três etapas: mouse laparotomia ventral, microinjeção transuterina, e<em&gt; In vivo</em&gt; Eletroporação. Nós usamos a microscopia de vídeo digital para demonstrar as críticas técnicas experimentais embriológicos.

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 ...

gene-transfer-to-developing-mouse-inner-ear-vivo

Research

JoVE Journal

Neuroscience

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

13.9K visualizações.  Oregon Health & Science University. A orelha interna do rato é um órgão sensorial placode derivado cujo programa é elaborado de desenvolvimento durante a gestação. Nós definimos uma In utero Técnica de transferência de gene consiste de três etapas: mouse laparotomia ventral, microinjeção transuterina, e In vivo Eletroporação. Nós usamos a microscopia de vídeo digital para demonstrar as críticas técnicas experimentais embriológicos.

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

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Entrega de genes ao cérebro pós-natal do rato por Non-ventricular Injection sHRNA e Eletroporação

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Neurociência

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

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

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

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

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 ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

Isolando-LacZ expressar Células de tecidos de camundongos Inner Ear usando Citometria de Fluxo

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

De alta resolução de imagem ao vivo de comportamento de células no desenvolvimento neuroepitélio

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

Gravações patch clamp em células ciliadas da orelha interna Isolado de Zebrafish

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

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 ...

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and genetic mechanisms that contribute to hearing loss. Introduction of vectors into the inner ear must be done in a way that widely distributes the agent throughout the cochlea while minimizing injury to the existing structures. This manuscript describes a post-auricular surgical approach that can be used for mouse cochlear therapy using molecular, pharmacologic, and viral delivery to mice postnatal day 10 and older via the round window membrane (RWM). This surgical approach enables rapid and direct delivery into the scala tympani while minimizing blood loss and avoiding animal mortality. This technique involves negligible or no damage to essential structures of the inner and middle ear as well as neck muscles while wholly preserving hearing. To demonstrate the efficacy of this surgical technique, the vesicular glutamate transporter 3 knockout (VGLUT3 KO) mice will be used as an example of a mouse model of congenital deafness that recovers hearing after delivery of VGLUT3 to the inner ear using an adeno-associated virus (AAV-1).

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

Método cirúrgico para Virally Mediated Gene entrega à orelha de rato Inner através da janela redonda Membrane

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...