Este trabalho foi financiado por doações da Fundação de Pesquisa para Surdez Yc S ea NSF (IOS 0930096) para KFB. KFB: NIH / NINDCD 2 RO1 DC04184, <a href="https://commons.era.nih.gov/commons/genericStatus.do?actionRole=nonPI&amp;applID=7854446&amp;uhf-token=Ugf7JvudZTCbTUW9jSAR1snMUgg%3D">3R01DC004184-08W1</a> , NSF DBI 0832862, 0930096 NSF IOS

1. Eletrodos para fazer Eletroporação <ol><li> Cortar 75 mM fio de tungstênio diâmetro (AM Systems, Inc. Carlsborg, WA) de comprimento adequado (cerca de 3-5 cm) </li><li> Coloque fio de tungstênio através de cabo conector molex (latão estampado pinos) </li><li> Enrole os fios de tungstênio e conector do cabo molex com tubulação do psiquiatra (SPC Technology, Chicago, IL) e aplicar calor com bico de Bunsen ou pistola de calor para selar o tubo de encolhimento para o fio </li><li> Para aguçar a ponta do fio de tungstênio, mergulhe o fio no hidróxido de sódio 1,0 N (Conrad et al. 1993) e, usando um clipe de papel como o outro eletrodo, electrolyze o fio com um Electroporator onda quadrada. As condições que usamos são 5 de 50 Volt pulsos cada uma com duração de 100 ms com 50 ms entre pulsos. Eletrodos deve ser aperfeiçoada para um diâmetro de 15-20 mM </li><li> Manter os fios em uma bandeja dentro de grooves pressionado em massa de modelar antes do uso em eletroporação </li></ol> 2. Set Up Station Eletroporação <ol><li> Fita afiada 75μm eléctrodos de tungsténio para micromanipuladores (instrumentos de precisão Mundial). </li><li> Micromanipuladores lugar em ambos os lados do palco estereomicroscópio (Leica). </li><li> Ligar os eléctrodos a um cuy-21 Editar Protect Praça Electroporator Wave (Figura 1). </li><li> Ligue o electroporator e estabelecer parâmetros para eletroporação, incluindo tensão, duração de pulso, e número de pulsos. Os parâmetros mais eficazes para este experimento foram encontrados para ser de três pulsos de 13 volts que cada durou 5.0ms, com 100ms entre cada pulso. Se eletroporação cria bolhas de ar no embrião, reduzir a duração do pulso. </li></ol> 3. Microinjeção de Morpholinos Em vesícula ótica Zebrafish <ol><li> Colheita de embriões zebrafish de um tanque de criação. Incubar os embriões em embrião de sensibilização média com 0,3 PPM de azul de metileno (Westerfield, 2005) com 28,5 ° C durante a noite. </li><li> Puxar agulhas de vidro com um puxador de Sutter P-97 eletrodo </li><li> Aquecer um gel de agarose base (1% agarose em água de peixe em um prato de 10 milímetros Petri) a 37 ° C em banho-maria </li><li> Heat 1% agarose de baixo ponto de fusão (IMPA) em peixes de água até derreter e mantê-lo aquecido em banho-maria a 37 ° C. </li><li> Preencher uma agulha de vidro com uma solução de morfolino (GeneTools, Corvallis, OR) e cortou a ponta de diâmetro adequado. Montar a agulha de injeção em um micromanipulador (Kuhn). </li><li> Cortar ponta da agulha para cerca de 10 mm e injetar o óleo mineral para garantir que a ponta permite a entrega suficiente de solução morfolino. </li><li> Dechorinate embriões em 24 postfertilization horas (hpf) e anestesiá-lo com MS222 (tricaina, Sigma) em 0,3 PPM peixes de água azul de metileno. </li><li> Alinhar 3-5 embriões anestesiados para a base de gel de agarose com o lado direito para cima para análise uniforme conveniente </li><li> Colocar 1-2 gotas de IMPA 1% sobre cada embrião e deixe solidificar para corrigir o embrião no lugar </li><li> Gire a placa de Petri, para que a vesícula ótica é no lado direito </li><li> Coloque a agulha dentro do lúmen da vesícula ótica e injetar a solução morfolino com um PV820 Pneumatic PicoPump (Instrumentos de Precisão World) com um tanque de nitrogênio em anexo (Figura 2) </li></ol> 4. Procedimento eletroporação <ol><li> Mover os embriões montado para a estação de eletroporação imediatamente após a injeção </li><li> Coloque o eletrodo positivo sobre o tecido imediatamente posterior à vesícula ótica, mas não penetrar; inserir o eletrodo negativo para o cérebro imediatamente anterior à vesícula ótica </li><li> Aplicar atual usando um pedal ou empurrando um interruptor. </li><li> Despeje a água de peixe em agarose e remover embriões de agarose com roda e uma pipeta de vidro. Tenha cuidado para não danificar os embriões. Para os embriões que são difíceis de remover, use uma agulha suavemente para romper qualquer IMPA em torno do embrião. </li><li> Embriões lugar em um prato com água peixe azul de metileno e elevar os embriões para estágios desejado para análise (morfológico, imuno-histoquímica, em anlaysis situ hybridization etc, e microscópica). </li></ol> 5. Resultados representativos: <img src="/files/ftp_upload/2466/2466fig1.jpg" alt="Figura 1" /> Figura 1. Estação de eletroporação. Eletrodos foram feitas usando fios de tungstênio afiadas 75μm e ligados por conduz a um cuy-21 Editar Protect Electroporator onda quadrada. Estes eletrodos foram gravadas para micromanipuladores. <img src="/files/ftp_upload/2466/2466fig2.jpg" alt="Figura 2" /> Figura 2. Estação de injeção. Todos os embriões foram injetados na orelha direita para fins de normalização. <img src="/files/ftp_upload/2466/2466fig3.jpg" alt="Figura 3" /> Figura 3. Coloração faloidina de filamentos de actina com 3 embriões dpf. (A) O embrião injecte 3 dpfd com controle de MO tem forte coloração de phalloidin na mácula posterior (pm). (B) O embrião que foi injetado com controle de MO e depois electroporated tinham níveis semelhantes de coloração em phalloidin pm nos embriões de injeção única. (C) Injeção com mif MOs na vesícula ótica sozinho não causou redução da coloração phalloidin, enquanto a injeção de mif MOs juntamente com eletroporação causou uma redução drástica de coloração phalloidin na pm (D). am mácula, anterior. <img src="/files/ftp_upload/2466/2466fig4.jpg" alt="Figura 4" /> Figura 4. Faloidina coloração dos filamentos de actina com 5 larvas dpf. Não houve diferença entre a injeção apenas (A) e injeção com eletroporação quando o padrão de controle de morfolino foi usado (B). No entanto, as larvas dpf 5 que foram injetados com mif morpholinos e electroporated mostraram redução da coloração phalloidin na mácula posterior (pm) (D), embora menos dramático do que três dpf, quando comparado com o embrião que foi injetado com mif morpholinos apenas (C ). am mácula, anterior. <img src="/files/ftp_upload/2466/2466fig5.jpg" alt="Figura 5" /> Figura 5. Coloração tubulina acetilados de embriões em 3 dpf. O embrião em (A) não recebeu qualquer injeção ou eletroporação. O embrião em (B) foi electroporated mas não recebeu a injeção. Embriões de receber a injeção e eletroporação (C, D) com mif morpholinos tinha visivelmente coloração tubulina diminuiu em comparação aos controles. (Macula pm, posterior). <img src="/files/ftp_upload/2466/2466fig6.jpg" alt="Figura 6" /> Figura 6. Coloração tubulina acetilados de embriões em 3 dpf com controle de morfolino. (A) Embrião sem nenhum tratamento mostrou forte coloração tubulina acetilada na mácula posterior (pm) e inervação extensiva. (B) Embrião injetados com controle de morfolino. (C) de embriões tratados com apenas eletroporação. (D) do embrião de controle com controle de morfolino injetado e electroporated tem níveis semelhantes de tubulina acetilada na mácula e inervação posterior comparação com o controle do tratamento não.

<ol>
	<li>Conrad, G. W., Bee, J. A., Roche, S. M., Teillet, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+microscalpels+by+electrolysis+of+tungsten+wire+in+a+meniscus.">Fabrication of microscalpels by electrolysis of tungsten wire in a meniscus.</a> J Neurosci Methods. 50, 123-127 (1993).</li><li>Suzuki, M., Takamura, Y., Maeno, M., Tochinai, S., Iyaguchi, D., Tanaka, I., Nishihira, J., Ishibashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+laevis+macrophage+migration+inhibitory+factor+is+essential+for+axis+formation+and+neural+development.">Xenopus laevis macrophage migration inhibitory factor is essential for axis formation and neural development.</a> J Biol Chem. 279, 21406-21414 (2004).</li><li>Barald, K. F., Kelley, M. W., W, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+placode+to+polarization:+new+tunes+in+inner+ear+development.">From placode to polarization: new tunes in inner ear development.</a> Development. 131, 4119-4135 (2004).</li></ol>

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Introduzimos com sucesso MOs oligonucleotídeo antisense no ouvido do embrião zebrafish 24 hpf. Embriões que foram levantadas após a injeção e eletroporação eram viáveis. Se os embriões sobreviveram à eletroporação, que cresceu a taxas normais em comparação com embriões que haviam recebido nenhum tratamento, bem como embriões que só tinha sido injetados e os embriões que só tinha sido electroporated. Observamos os efeitos da mif e mif-like MOs após a injeção de ouvido e eletroporação através da rotulagem com phalloidin e tubulina acetilados. Faloidina coloração dos filamentos de actina em células sensoriais ciliadas da mácula posterior indica que eletroporação de mif e mif-like MO na fase 24 hpf causou mudanças na célula de cabelo e / ou números de estereocílios, quando comparado aos embriões que foram apenas injetado com o MOs (Figura 3). Esta diminuição no número de células ciliadas é consistente com os achados de Shen et al. (Submetido) que MOs para mif e mif-como causar uma redução no número de células ciliadas da mácula sacular. A diminuição do número de células de cabelo em embriões que foram injetados com MOs e electroporated demonstrar que eletroporação pode ajudar em mover o MOs através da membrana celular, aumentando assim a extensão dos efeitos morfológicas quando comparadas com embriões no qual a vesícula ótica só foi injetado. Alterações na morfologia do gânglio statoacoustic foram observados através de coloração tubulina acetilados. A extensão da inervação pelo gânglio statoacoustic foi afetada, como embriões que foram injetados e electroporated mostram diminuição da ramificação das neurites (Figura 5). Também pode haver diminuição da condensação das células ganglionares e / ou diminuição do número de células ganglionares, porque a coloração tubulina acetilada é significativamente diminuída em embriões que eram injetadas e electroporated. Mif porque tem se mostrado fundamental para o desenvolvimento do sistema nervoso (Suzuki et al., 2004), as mudanças observadas após a eletroporação pode ser o resultado de transfecção aumento da mif e mif-MO como solução para as células neuroblastos. Eletroporação de oligo antisense morpholinos diretamente em um tecido em desenvolvimento é uma ferramenta útil para controlar a expressão de um gene durante o desenvolvimento que o tecido, tanto espacialmente e temporalmente, no embrião. Eletroporação de mif e mif-like MOs para os tecidos do ouvido interno resultou em alterações morfológicas de ambos macula o posterior eo gânglio statoacoustic. Houve uma redução no número de células sensoriais ciliadas na mácula posterior nos embriões que haviam sido injetados e electroporated em comparação com embriões que haviam recebido nenhum tratamento ou com embriões que tinham sido apenas injetados ou apenas electroporated. Houve também uma diminuição no grau de inervação do patch posterior sensoriais pelo gânglio statoacoustic eo tamanho do SAG. Eletroporação é um método valioso para transfecting macromoléculas em tecidos específicos, com a vantagem de observar os efeitos primários do que o DNA particular, RNA, ou MO no tecido desejado e discriminar estes de efeitos secundários sobre outros tecidos, incluindo o cérebro, da crista neural e mesênquima periotic sobre o desenvolvimento do ouvido interno (Barald e Kelley, 2004).

<ol><li> MOs: 0,3 mM de uma combinação de para mif, mif-like MO1 e mif-like MO2 foi usado. Mif MO é gratuito para o códon de iniciação do mRNA zebrafish mif: 5&#39;-acatcggcatgactgcgacagagat-3 &#39;. Dois MOs foram usados ​​para mif-like mif-like MO1 é gratuito para o local de início de translação:. 5&#39;-GTTTCTATATTTATGAACGGCATGA-3 &#39;, enquanto mif-like MO2 derivado AS seqüência na fronteira intron1/exon 2: 5&#39;-GATTCATCCTCTGAAGACGTAAGCC -3 &#39;. Controle de MO foi a seqüência de nucleotídeos a partir de Ferramentas mexidos Gene: 5&#39;-CCTCTTACCTCAGTTACAATTTATA-3 &#39;. </li><li> azul de metileno (0,3 ppm) em água de peixes </li><li> MS222 (tricaina, Sigma, 0,64 mM em água de peixe) </li><li> Baixo ponto de fusão de agarose (IMPA; Roche) </li><li> vermelho de fenol (Sigma) </li><li> Anti-acetilado tubulina (Sigma) </li><li> Alexa Fluor ® 488 cabras de anticorpos anti-rato segundo (sondas Molecular) </li><li> Texas vermelho-phalloidin (sondas Molecular) </li></ol>

direct delivery of mif morpholinos into the zebrafish otocyst by injection and electroporation affects inner ear development

Nos últimos anos, eletroporação tornou-se uma técnica popular no transfecção in vivo de DNA, RNA e morpholinos em vários tecidos, incluindo o olho, cérebro e somitos do peixe-zebra. A vantagem de eletroporação sobre outros métodos de manipulação genética é que tecidos específicos podem ser direcionados, tanto espacialmente e temporalmente, para a introdução de macromoléculas pela aplicação de corrente elétrica. Aqui nós descrevemos o uso da eletroporação para transfecting mif e mif-like morpholinos para os tecidos do ouvido desenvolvimento interno do zebrafish. Em estudos anteriores, mif morfolino injetadas em embriões no 1 - de 8 células estágio resultou em ampla alterações morfológicas no sistema nervoso e olho, assim como o ouvido. Ao direcionar os tecidos do ouvido interno em fases posteriores de desenvolvimento, podemos determinar os efeitos primários da MIF no ouvido desenvolvimento interior, em oposição a efeitos secundários que podem resultar da influência de outros tecidos. Usando tubulina phalloidin e acetilada coloração para estudar a morfologia de neurônios, processos neuronais e células ciliadas da mácula associado a posterior, fomos capazes de avaliar a eficácia da eletroporação como um método de transfecção alvejado no ouvido zebrafish interior. As vesículas ótica de embriões foram injetados com 24hpf morpholinos e electroporated e foram comparados com embriões que haviam recebido nenhum tratamento ou tinham sido injetados ou apenas electroporated. Embriões que foram injetados e electroporated mostraram uma diminuição no número de células de cabelo, diminuição da inervação pelo gânglio statoacoustic (SAG) e menos neurônios SAG comparados com grupos controle. Nossos resultados mostraram que a entrega direta de morpholinos em otocysts em fases posteriores evita o sistema nervoso não-específica e efeitos da crista neural de morpholinos emitidos na fase celular 1-8. Ele também permite o exame dos efeitos que são direcionados para o ouvido interno e não efeitos secundários sobre a orelha de efeitos primários sobre o cérebro, ou mesênquima da crista neural periotic.

Watch this Scientific Journal Video about Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development at JoVE.com

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

Um método para entregar morpholinos diretamente no otocyst zebrafish em 24hpf tem sido desenvolvido. Usando microinjeção de morpholinos no lúmen da vesícula ótica e eletroporação para efeito de penetração, fomos capazes de contornar o efeito de morpholinos sobre o cérebro e obter efeitos específicos para o ouvido interno.

Entrega direta da MIF Morpholinos Into the Otocyst Zebrafish por injeção e Eletroporação afeta o desenvolvimento da orelha interna

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the ...

direct-delivery-mif-morpholinos-into-zebrafish-otocyst-injection

Research

JoVE Journal

Biology

14.2K visualizações.  University of Wisconsin, Madison. Um método para entregar morpholinos diretamente no otocyst zebrafish em 24hpf tem sido desenvolvido. Usando microinjeção de morpholinos no lúmen da vesícula ótica e eletroporação para efeito de penetração, fomos capazes de contornar o efeito de morpholinos sobre o cérebro e obter efeitos específicos para o ouvido interno.

Vídeo: Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

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

In Utero injeção intraventricular e eletroporação de embriões de camundongos E15

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

Biologia

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

Dissecação bruta e Multa de Ouvido Interno Epitélio sensorial em adultos Zebrafish ( Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

Cultura primária e Eletroporação Plasmid do Órgão de Corti murino.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

Injeção intraperitoneal em Zebrafish Adulto

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

RNAi Interferência por injeção dsRNA em Drosophila Os embriões

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

Eletroporação de Mesênquima Craniofacial

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

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 ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

Transferência de Embriões em Gene de frango por mais velhos<em&gt; Ovo ex</em&gt; Eletroporação

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. The first procedure consists of perforation of the tympanic bulla, termed bullostomy; the second one is a transtympanic injection. Both emulate human clinical intratympanic procedures.
Chitosan-glycerophosphate (CGP) and Ringer&#180;s Lactate buffer (RL) were used as biocompatible vehicles for local drug delivery. CGP is a nontoxic biodegradable polymer widely used in pharmaceutical applications. It is a viscous liquid at RT&#160;but it congeals to a semi solid phase at body temperature. RL is an isotonic solution used for intravenous administrations in humans. A small volume of this vehicle is precisely placed on the Round Window (RW) niche by means of a bullostomy. A transtympanic injection fills the middle ear and allows less control but broader access to the inner ear.
The safety profiles of both techniques were studied and compared by using functional and morphological tests. Hearing was evaluated by registering the Auditory Brainstem Response (ABR) before and several times after microsurgery. The cytoarchitecture and preservation level of cochlear structures were studied by conventional histological techniques in paraformaldehyde-fixed and decalcified cochlear samples. In parallel, unfixed cochlear samples were taken and immediately frozen to analyze gene expression profiles of inflammatory markers by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR).
Both procedures are suitable as drug delivery methods into the mouse middle ear, although transtympanic injection proved to be less invasive compared to bullostomy.

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

Two microsurgery approaches for local drug delivery to the inner ear are described here and compared in terms of impact on hearing parameters, cochlear cytoarchitecture and expression of inflammatory markers.

Um Estudo Comparativo de drogas métodos de entrega direcionados à orelha de rato Inner: Bullostomy<em&gt; Versus</em&gt; Injeção transtimpânica

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. ...