Os autores agradecem Sharmin Hossain para o crescimento de filmes time-lapse captura de um neurônio imaturo e Dunfield Derek para as imagens de microscopia eletrônica de dicas SCE micromicropipette.

Equipamentos de set-up: <ol><li> Os equipamentos elétricos necessários para esta técnica são um estimulador, um osciloscópio, e um suporte de micropipeta equipado com um fio de prata ao ser inserido na micropipeta. Normalmente usamos uma Axon instrumentos Axoporator 800A estimulador, no entanto outros estimuladores comuns, tais como os instrumentos estimuladores Grama SD9 também são adequados para uma única célula eletroporação. O estimulador é ligado ao headstage que faz interface com o eletrodo de arame de prata para ser colocado dentro da micropipeta de vidro contendo uma solução interna de condução. Outro fio do estimulador é ligado à entrada do osciloscópio, que também recebe a entrada do chão girino externo. O chão girino externa é um simples fio de prata que permanece em contato com o electrotonic girino durante a eletroporação. O circuito está completo uma vez que a micropipeta é colocado em contato com o girino. </li><li> Eletroporação única célula exige um microscópio com uma boa distância de trabalho (pelo menos 5 centímetros), pois isto dá margem de manobra e permite que o micropipeta para ser trazido em um ângulo de cerca de 30-45 graus. </li></ol> Fabricação de micropipetas: Preparação de micropipetas adequado é um passo crítico neste protocolo. A dificuldade reside em equilibrar uma ponta estreita (menos de 1 Hm de diâmetro) com um que não vai quebrar facilmente quando a perfuração do tecido. Temos também descobriram que as pontas com um ângulo de inclinação de mais de 10 graus são preferíveis. <ol><li> Prepare uma micropipeta de vidro puxado com um diâmetro da ponta de menos de 1 Hm, e um ângulo de ponta do cone maior que 10 graus. <ol><li> Vidro de borosilicato com um filamento interna com diâmetro externo de diâmetro 1,5 mm e interno de 0,75 milímetros é bem adequado para este fim. <ol><li> O vidro relativamente grossa fornece um grau de rigidez à ponta. </li><li> O filamento interno é importante na elaboração de volta cheio de líquido para a ponta micropipeta. </li></ol></li></ol></li><li> Fabricação de dicas adequadas, muitas vezes, exigem várias modificações com base nos resultados observados. Diferentes tecidos pode exigir uma ponta com uma forma ligeiramente diferente ou diâmetro da ponta. </li></ol> De uma única célula protocolo eletroporação: Para novos usuários, é aconselhável começar com corantes fluorescentes dextran uma vez que, ao contrário geneticamente codificado proteínas fluorescentes, que pode ser imediatamente visto sob epifluorescência. Uso de corantes fluorescentes permite ao usuário ver diretamente a localização da ponta da micropipeta dentro do tecido, e sobre eletroporação dá um feedback instantâneo para saber se o equipamento foi configurado corretamente e se a ponta micropipeta é apropriado. <ol><li> Preencha micropipeta com solução do composto a ser electroporated ou usando uma seringa de carga ou de back-enchimento. <ol><li> Se estiver usando DNA plasmídeo, garantir que as amostras são-endotoxina livre, e em uma concentração entre 1-2μg/ml, preparada em água. </li><li> A concentração de corantes fluorescentes dextran deve ser determinado empiricamente. Muitas vezes usamos corantes fluorescentes diluído em água até cerca de 300μM. </li><li> Normalmente, os volumes de 0,5-1μl são suficientes. </li><li> Antes do carregamento, brevemente centrífuga soluções em alta velocidade para reduzir a quantidade de detritos de partículas atraídas para a micropipeta, que pode levar ao entupimento. </li><li> Bolhas de ar dentro da ponta, muitas vezes afetam o fluxo de corrente e deve ser desalojado por flicking vigorosamente a micropipeta antes da montagem na headstage. </li></ol></li><li> Inserir micropipeta para titular ou headstage montado em um micromanipulador de 3 eixos. Assegurar que os eletrodos fio de prata é inserido micropipeta, e está em contato com a solução electrotonic interna. </li><li> Anestesiar girinos por imersão em 0,02% MS-222 (3-aminobenzóico ácido etil éster) preparada em meio de criação de girinos (solução Steinberg, pH 7,4). <ol><li> Normalmente usamos estágio 44-48 albino Xenopus laevis girinos em nossos experimentos. </li><li> Girinos são geralmente totalmente anestesiado após aproximadamente 5 minutos. </li><li> Girinos podem ser anestesiados várias ao mesmo tempo para reduzir tempo de espera, no entanto, evitar a exposição a MS-222, por períodos superiores a 1 hora. </li></ol></li><li> Transferência de um girino única anestesiados para a câmara de eletroporação usando uma micropipeta transferência de plástico. <ol><li> A câmara de eletroporação pode ser feita facilmente em casa por esculpir uma cavidade em forma de girino em uma pequena Sylgard ® bloco de silicone ea inserção de um fio terra de prata na cavidade tal que estarão em contato com o girino electrotonic na câmara. </li></ol></li><li> Posição dorsal lado girino até usando um pincel umedecido. </li><li> Abaixe a micropipeta até que esteja em contacto com a pele, e adjacente à região de interesse. <ol><li> Segmentação regiões com corpos celulares densamente vai aumentar muito as chances de sucesso eletroporação.Normalmente, o alvo tectum girino óptica. </li><li> Trazer a micropipeta em um ângulo de 30 a 45 graus. Trajetórias mais rasas fazer cortes na pele mais difícil, enquanto os ângulos mais íngremes, muitas vezes obstruem nossa visão. </li></ol></li><li> Redução contínua da micropipeta inicialmente dimple a pele, e depois passar para o tecido subjacente. <ol><li> Células superficiais são os preferidos para in vivo de imagens, e pode ser alvo de garantir que a ponta micropipeta é reduzida lentamente sobre ondulações da pele. </li></ol></li><li> Deve-se monitorar constantemente a resistência micropipeta uma vez colocado dentro do tecido. Se estiver usando uma 800A Axoporator (Axon Instruments), resistências de aproximadamente 10 40MΩ são ótimas. A resistência é um bom indicador de se o diâmetro da ponta é apropriado para uma única célula eletroporação. <ol><li> Resistências micropipeta alta (&gt; 100MΩ) são indicativos de dicas entupidos ou micropipetas com dicas que são muito estreitas. </li><li> Resistências micropipeta baixo (&lt;10MΩ) são indicativos de um diâmetro da ponta que é muito grande, muitas vezes resultado de uma ponta quebrada. </li><li> Se estiver usando um estimulador que não fornece uma medida direta da resistência, a resistência micropipeta pode indirectamente medidos, observando a amplitude dos pulsos de voltagem medida pelo osciloscópio (discutido abaixo). </li></ol></li><li> Aplicar trem de tensão. <ol><li> Para DNA plasmídeo, pulso trens encontrado para ser mais eficazes consistem em pulsos de onda quadrada negativa 1ms de duração, entregue em 300Hz com duração de pulso de trem de 500ms. <ol><li> A tensão do pulso é determinada empiricamente tal que a amplitude de pulsos medidos no osciloscópio é entre 0,75 e 1.5μA. </li></ol></li><li> Para corantes fluorescentes dextran, positiva pulsos de onda quadrada de duração 300μs são entregues em uma freqüência de 300Hz com duração de pulso de trem de 10ms. <ol><li> Como com o protocolo de DNA, a tensão do pulso é determinada empiricamente tal que a amplitude de pulsos medidos no osciloscópio é entre 0,75 e 1.5μA. </li><li> Ajustes adicionais para os parâmetros de estímulo pode ser feito com base na observação dos resultados de eletroporação sob epifluorescência. <ol><li> Se as células electroporated aparecem dim, os parâmetros de pulso deve ser gradualmente aumentada até que as células aparecem mais brilhantes. </li><li> Por outro lado, se grupos de células são rotulados, os parâmetros de pulso deve ser reduzida até células individuais são rotulados. </li></ol></li></ol></li></ol></li><li> Retrair micropipeta, re-inserir em um site diferente dentro do tecido, e aplicar trem de tensão. <ol><li> Para melhorar o rendimento, que normalmente electroporate vários sites em cada hemisfério do tectum girino óptica com espaçamento suficiente para assegurar que células marcadas não se sobrepõem. </li></ol></li><li> Transferir o girino electroporated em um recipiente com solução de Steinberg criação. <ol><li> Girinos se recuperar rapidamente da anestesia em poucos minutos. </li></ol></li><li> A micropipeta mesmo pode ser usado para girinos numerosos. É importante observar constantemente a amplitude dos pulsos observados no osciloscópio. <ol><li> Se a amplitude do pulso permanece baixa após a aumentar significativamente a intensidade de pulso este é provavelmente um resultado da ponta sendo obstruídas, muitas vezes visto quando electroporating muitos girinos em uma sessão. <ol><li> Um método para desalojar uma obstrução ponta menor é a aplicação de um único pulso positivo depois de inserir o micropipeta para o tecido. </li><li> Se a obstrução não pode ser desalojado, ou entupimento recorrência freqüente, substitua a micropipeta. </li></ol></li><li> Se a amplitude do pulso continua a ser muito grande após diminuindo significativamente os parâmetros de pulso, este é um sinal de que a ponta quebrou e precisa ser substituído. </li></ol></li></ol> Triagem para as células com sucesso electroporated: <ol><li> Após a eletroporação, os girinos são selecionados para células marcadas usando um microscópio de epifluorescência, na posição vertical. </li><li> No caso de tinturas fluorescentes, os girinos podem ser rastreados após tão pouco como 30 minutos após a eletroporação. <ol><li> Encontramos, no entanto, que intervalos mais longos estão associados com níveis mais baixos de fluorescência de fundo. </li></ol></li><li> Para geneticamente codificado proteínas fluorescentes, a triagem é geralmente conduzida pelo menos 12 horas após a eletroporação. <ol><li> Células que expressam proteínas fluorescentes continuará a receber mais brilhantes com o tempo, portanto, células dim pode ser re-examinados após um intervalo adicional de 12-24 horas. </li></ol></li><li> Girinos são anestesiados, como descrito acima, colocadas em uma câmara de Sylgard ® e lamínulas. Girinos individuais são, então, examinado sob epifluorescência. <ol><li> Note-se que a exposição excessiva à luz de epifluorescência, irá resultar em fototoxicidade que pode matar a célula rotulados. Portanto, minimizar a quantidade de tempo que as células são expostas à luz fluorescente. </li></ol></li><li> Retorno girinos para recipiente com solução de Steinberg criação. </li></ol>

<ol>
	<li>Bestman, J. E., Ewald, R. C., Chiu, S. L., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+single-cell+electroporation+for+transfer+of+DNA+and+macromolecules.">In vivo single-cell electroporation for transfer of DNA and macromolecules.</a> Nature Protocols. 1, 1267-1272 (2006).</li><li>Dunfield, D., Haas, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+electroporation.">Single cell electroporation.</a> Encyclopedia of Neuroscience (4th Ed.). , .</li><li>Haas, K., Jensen, K., Sin, W. C., Foa, L., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+electroporation+in+Xenopus+tadpoles+in+vivo--from+single+cells+to+the+entire+brain.">Targeted electroporation in Xenopus tadpoles in vivo--from single cells to the entire brain.</a> Differentiation. 70, 148-154 (2002).</li><li>Haas, K., Sin, W. C., Javaherian, A., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+electroporation+for+gene+transfer+in+vivo.">Single-cell electroporation for gene transfer in vivo.</a> Neuron. 29, 583-591 (2001).</li></ol>

De uma única célula eletroporação (SCE) é uma ferramenta poderosa para determinar a função do gene e realizando manipulação genética alvo. A transparência do girino albino e da acessibilidade do cérebro tornam este sistema modelo ideal para a visualização de crescimento neuronal e eventos intracelulares dentro de um organismo vivo. SCE permite a visualização do crescimento de um único neurônio, e para realizar manipulações de células-autônoma dentro de um cérebro de outra forma inalterada. Embora este artigo vídeo de...

single cell electroporation in vivo within the intact developing brain

De uma única célula eletroporação (SCE) é uma técnica especializada que permite a entrega de DNA ou outras macromoléculas em células individuais dentro do tecido intacto, inclusive em preparações in vivo. A vantagem desta técnica é que manipulações experimentais podem ser realizadas em células individuais, deixando o tecido circundante inalterada, assim, distinguir células autônomas efeitos daqueles decorrentes de tratamentos global. Quando combinado com o avançado em técnicas de imagem in vivo, a SCE de marcadores fluorescentes permite a visualização direta da morfologia celular, crescimento celular, e eventos intracelulares em escalas de tempo variando de segundos a dias. Embora esta técnica é usada em uma variedade de preparações in vivo e ex vivo, temos esta técnica otimizada para uso em girinos Xenopus laevis. Neste artigo de vídeo, detalhamos o procedimento para a SCE de um corante fluorescente ou DNA plasmídeo em neurônios dentro do cérebro intacto do albino Xenopus girino. Discutimos, também, métodos para otimizar o rendimento, e mostrar exemplos de imagens de dois fótons de fluorescência de neurônios ao vivo fluorescente etiquetado pelo SCE.

Watch this Scientific Journal Video about Single Cell Electroporation in vivo within the Intact Developing Brain at JoVE.com

Single Cell Electroporation in vivo within the Intact Developing Brain

De uma única célula eletroporação (SCE) é uma técnica especializada permitindo a entrega de DNA ou outras macromoléculas em células individuais dentro do tecido intacto, inclusive em preparações in vivo. Aqui detalhamos o procedimento para a SCE de um corante fluorescente ou DNA plasmídeo em neurônios dentro do cérebro intacto do Xenopus laevis girino.

Eletroporação única célula in vivo dentro do cérebro intacto desenvolvimento

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

single-cell-electroporation-in-vivo-within-the-intact-developing-brain

Research

JoVE Journal

Biology

13.2K visualizações.  University of British Columbia - UBC. De uma única célula eletroporação (SCE) é uma técnica especializada permitindo a entrega de DNA ou outras macromoléculas em células individuais dentro do tecido intacto, inclusive em preparações in vivo. Aqui detalhamos o procedimento para a SCE de um corante fluorescente ou DNA plasmídeo em neurônios dentro do cérebro intacto do Xenopus laevis girino.

Vídeo: Single Cell Electroporation in vivo within the Intact Developing Brain

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

Eletroporação de Micobactérias

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

Biologia

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

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

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

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

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

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

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

Transfecção de DNA de mamíferos usando músculos esqueléticos In Vivo Eletroporação

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Gravações de uma única célula de sucção de cones fotorreceptores rato

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

Na imagem in vivo de larvas Drosophila Intact no Sub-celular Resolução

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Em Eletroporação vivo de Morpholinos na Retina Zebrafish Adulto

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

Na eletroporação in vivo de Morpholinos no Adulto Regeneração Zebrafish Cauda Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

Imagem confocal de Solteiro mitocondriais superóxido Pisca em coração intacto ou<em&gt; In Vivo</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Avaliação do cálcio Faíscas intactas fibras musculares esqueléticas

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

In Vivo Microinjection and Electroporation of Mouse Testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis.
The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV).
For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular&#160;cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red&#160;fluorescence, illustrating transfection success.
Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.

In Vivo Microinjection and Electroporation of Mouse Testis

This article describes microinjection and electroporation of mouse testis in&#160;vivo as a&#160;transfection technique for testicular mouse cells to study unique processes of spermatogenesis. The presented protocol involves steps of glass capillary preparation, microinjection via the efferent duct, and transfection by electroporation.

In Vivo microinjeção e Eletroporação de testículos de camundongo

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...