Gostaríamos de agradecer ao Sr. Maaz Enver para o uso de sua câmera HD (Cannon VIXIA HFS100). Este trabalho foi apoiado em parte por concessões do NIH (EY018738), Nova Jersey, Comissão de Pesquisa da Medula Espinhal (08-3074-SCR-E-0 e 10-3091-SCR-E-0), e Prêmio de Pesquisa Biomédica Busch.

Uma vez que algumas partes do protocolo são realizados conforme descrito anteriormente em outros vídeos Jové 2 e papéis 3 com modificações menores não vamos discutir aqueles em detalhe aqui. 1. Manuseio de ovo e Preparação Agulha <ol><li> Os ovos podem ser armazenados em um refrigerador de vinho menos cerca de 13 ° C por até 1 semana. Se a temperatura é demasiado elevada, os embriões vai começar a desenvolver de forma anormal, enquanto que, baixa temperatura provoca uma mortalidade de alta. Quando estiver pronto para incubar tirar os ovos do refrigerador de vinho e definir os ovos na vertical com a extremidade maior até. Mantenha os ovos em a temperatura da sala durante pelo menos 2 horas antes de colocá-los no 37,5 ° C incubadora. </li><li> Incubam os ovos para 96-100 horas, que é sobre o dia embrionário 4 para obter embriões que estão na fase Hamburger e Hamilton 22 4-5. </li><li> Prepare agulhas micropipeta de tubos de vidro puxado capilares e quebrar a ponta em uma micro dissecaçãoescopo com uma pinça para conseguir uma abertura cerca de 0,1 m de diâmetro e um cone de 20 mm. Agulhas com pontas maiores têm dificuldade perfurar a membrana vitelina enquanto pontas menores têm dificuldade de carregar e entregar a solução de DNA. </li><li> Anexar a agulha para um 0,1 ml Hamilton estanque ao gás seringa montada sobre um micromanipulador. Use um pequeno pedaço de Masterplex tubo de silicone para anexar a agulha para a seringa. Uma vez que as conexões estão apertadas não há necessidade de adicionar óleo mineral para selar este anexo. </li><li> Misture 2 uL de repórter solução de ADN plasmídeo com uma concentração que varia 3-6 ug / uL e 0,2 uL de verde rápido (0,025%) em um pedaço de parafilme. Corante verde rápido vai ajudar a visualizar a injeção. Lentamente, carregar a agulha com a mistura. </li><li> Para liberar a membrana vitelina da membrana interna girar o ovo suavemente cerca de 180 ° e esperar por alguns minutos, em seguida, gire-o de volta à posição original e configurá-lo para eletroporação. </li><li> Limpe a forceps e casca de ovo com etanol a 70% para evitar a infecção para o embrião. </li><li> Faça um pequeno buraco sobre o ovo imediatamente acima da célula de ar com um par de fórceps tamanho AA. Tenha cuidado para não quebrar a casca do ovo. Remover pedaços pequenos um de cada vez para fazer uma pequena janela. Cuidadosamente remova a membrana interna usando os fórceps sem tocar a membrana vitelina. </li></ol> 2. Injeção e Eletroporação <ol><li> Para evitar danos para o cérebro ou o coração posição, o contra-agulha lateral para o bundle principal de vasos sanguíneos que entram no olho e apontando em direção ao bico. </li><li> Pierce através da membrana vitelina, esclera, retina e humor vítreo por um impulso súbito suave da agulha. Se a agulha perfura através de a outra extremidade do olho que deve ser alright, a menos que ele danifica qualquer veia sangue major. </li><li> Lentamente, puxe de volta a agulha na borda da abertura e colocá-lo quase tangente à parede exterior do globo ocular. </li><li> Insert a agulha para dentro do espaço sub da retina entre a esclera e da retina. </li><li> Injectar o DNA até que você possa visualizar a solução verde de enchimento o lado do globo ocular e empurrando a fressura da retina através da criação de um bojo. </li><li> Se a sua colocação da agulha não é correto, então você vai ver a solução de DNA se espalhando dentro do humor vítreo enchendo até o meio do globo ocular. Além disso, se você danificar a retina muito, então você vai ver a solução de DNA está saindo do globo ocular. </li><li> Lentamente, retirar a agulha e coloque imediatamente os eletrodos em paralelo dentro do ovo após imersão em PBS. Empurre os eletrodos para submergir no líquido amniótico de uma forma para que o olho injetado está localizado entre os eletrodos. Evite tocar em qualquer grande vaso sanguíneo ou o coração com os eletrodos, enquanto colocá-los. O eletrodo negativo deve ser na parte lateral da injecção para que o DNA pode ser transportados a partir do espaço da retina sub para a retina em direção eletrodo positivo. Electrocorporar a retina com 5 pulsos de 15V para 50 ms com intervalos de 950 ms. </li><li> Com cuidado, retire os eletrodos e selar a janela do ovo com pedaços de fita adesiva transparente. </li><li> Etiqueta e datar o ovo injetado antes de colocá-lo de volta para a incubadora. Tipicamente, ele leva cerca de 3 a 5 minutos para completar o processo de electroporação todo. </li><li> Expressão da GFP pode ser visto tão cedo quanto 8 horas após a electroporação. No entanto, você pode esperar até que o embrião atinge a fase desejada antes da colheita. </li></ol> 3. Os resultados representativos Em nosso estudo, nós usamos vários construtos plasmídicos para estudar a regulação da expressão de gene que envolveu o desenvolvimento de células de retina. Neste vídeo pCAG-GFP (controle de transfecção) foi usado para siga uma injecção de bem-sucedida e electroporação. No entanto, qualquer constructo plasmídeo com gene repórter (GFP, RFP etc) pode ser usado. Mesmo embora expressão da GFP pode ser visto tão cedo quanto 8 horas após a elemenctroporation, que normalmente começar a colher o óvulo no dia 6 (E6) e em diante. Eletroporados retinas foram dissecadas para fora do embrião e analisados ​​em microscópio fluorescente dissecção antes de incorporação e de corte. Tipicamente, expressão do gene repórter pode ser visto, pelo menos, em um quarto da retina após uma electroporação bem-sucedida (Fig. 1). Os tecidos da retina transfectadas foram analisados ​​por meio de seccionamento de uma clara visualização de morfologias celulares. A imuno-histoquímica utilizando marcadores de células tipo específico (Brn3a, Pax6 etc) de caracterização permitido de célula-específica expressão da GFP (Fig 2). <img src="/files/ftp_upload/3792/3792fig1.jpg" align="Figure 1" /> Figura 1. Eletroporação sucesso de expressão plasmídeo repórter GFP resultados positivos. Chicken embrionário retinas foram injetados e electroporados no dia embrionário 4 e colhidas no dia embrionário 6. , Pelo menos, 25% da retina com êxito foi transfectado (Um modo de exibição = Top, B= View Inferior). Escala Bar = 1mm. <img src="/files/ftp_upload/3792/3792fig2.jpg" align="Figure 2" /> Figura 2. Caracterização de células da retina expressando GFP utilizando imuno-histoquímica. GFP expressar tecidos da retina em E7 foram palco fixo e seccionados. Estas secções foram, então, corados com marcadores de células vários específicas. Brn3a foi utilizado para determinar células ganglionares (A) enquanto Pax6 foi utilizado para determinar amacrina, horizontal e células ganglionares (B). Barra de escala = 50 mM.

<ol>
	<li>Doh, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+retinal+cell+development+in+chick+embryo+by+immunohistochemistry+and+in+ovo+electroporation+techniques.">Analysis of retinal cell development in chick embryo by immunohistochemistry and in ovo electroporation techniques.</a> BMC. Dev. Biol. 10, (2010).</li><li>Blank, M. C., Chizhikov, V., Millen, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Ovo+Electroporations+of+HH+Stage+10+Chicken+Embryos.">In Ovo Electroporations of HH Stage 10 Chicken Embryos.</a> J. Vis. Exp. (9), e408-e408 (2007).</li><li>Nakamura, H., Funahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+DNA+into+chick+embryos+by+in+ovo+electroporation.">Introduction of DNA into chick embryos by in ovo electroporation.</a> Methods. 24, 43-48 (2001).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.+1951.">A series of normal stages in the development of the chick embryo. 1951.</a> Dev. Dyn.. 195, 231-272 (1992).</li><li>Hamburger, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stage+series+of+the+chick+embryo.">The stage series of the chick embryo.</a> Dev. Dyn. 195, 273-275 (1992).</li></ol>

Todos os experimentos com animais foram aprovados pelo Institutional Animal Care e do Comitê Instalações da Universidade Rutgers.

Targeted injecção da retina e em eletroporação in ovo em retina embrionária no estádio de E4 podem objetivar especificamente as células progenitoras da retina, resultando em a habilidade de visualizar todos os seis principais tipos de células da retina no nível da célula única. Em electroporação in ovo em HH10 (~ E1.5) alvejando o vesícula óptica é capaz de transfectar células que se desenvolvem para formar o olho. No entanto, estas células têm um volume de negócio...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Fertilizados livres de patógenos (SPF) de frango ovos brancos leghorn </td><td> Fazendas Sunrise (Catskill, Nova Iorque) </td><td></td><td></td></tr><tr><td> Chicken ovo incubadora </td><td> GQF fabricação, Savannah, GA </td><td> Modelo-1550 </td><td> Definir a 60% de umidade e 37,5 ° C. </td></tr><tr><td> Os tubos de vidro capilares </td><td> Precision Mundial Instrument Inc. </td><td> TW150F-4 </td><td></td></tr><tr><td> 0,1 ml seringa </td><td> Hamilton Co. Reno, Nevada </td><td> Estanques 1710 </td><td> Seringa Alternativamente 1ml pode ser usado como bem </td></tr><tr><td> Masterflex Silicone (peróxido) Tubulação </td><td> Cole-Parmer </td><td> HV-96.400-13 </td&gt; <td> Corte um pedaço pequeno (1 cm) para anexar a agulha de vidro para a seringa </td></tr><tr><td> Pinça </td><td> Dumont </td><td> AA </td><td></td></tr><tr><td> Micromanipulador </td><td> Precision Mundial Instrument Inc. </td><td> M3301-M3 </td><td></td></tr><tr><td> O DNA de plasmídeo </td><td></td><td></td><td> pCAG-GFP foi emprestado do Dr. Connie Cepko </td></tr><tr><td> Fast Green FCF </td><td> Sigma-Aldrich </td><td> F7252 </td><td> Diluir-lo para 0,025% com PBS </td></tr><tr><td> Gerador de pulso </td><td> Harvard Apparatus, MA </td><td> BTX ECM 830 </td><td> Gerador de onda quadrada </td></tr><tr><td> Eletrodos </td><td> Harvard Apparatus, MA </td><td> BTX modelo 514 </td><td> Nossos eletrodos foram espaçadas 3-5 mm além </td></tr><tr><td> Câmera Digital Monocromático </td><td> Zeiss, Alemanha </td><td> Axiocam MRM </td><td></td></tr><tr><td> Microscópio de dissecção fluorescente </td><td> Leica Microsystems, Alemanha </td><td> Leica MZ16FA </td><td></td></tr><tr><td> Microscópio de fluorescência Upright </td><td> Zeiss, Alemanha </td><td> Zeiss Axio Imager A1 </td><td></td></tr></tbody></table>

in ovo electroporation in embryonic chick retina

Retina embrionária de galinha é uma excelente ferramenta para estudar o desenvolvimento da retina em vertebrados mais elevados. Por causa de grande tamanho e desenvolvimento externo, ele é comparativamente muito fácil de manipular a retina de pinto embrionário usando recombinante DNA / tecnologia de RNA. Electroporação de ADN / ARN construtos para a retina embrionária têm uma grande vantagem para estudar regulação do gene em retinianas-tronco / progenitoras células durante o desenvolvimento da retina. Tipo diferente de ensaios, tais como ensaio de repórter gene, gene over-expressão, gene knock down (shRNA), etc pode ser realizada utilizando a técnica de electroporação. Este vídeo demonstra alvejado injecção da retina e em eletroporação in ovo dentro da retina de pinto embrionário no Hamburger e do estágio Hamilton 22-23, o que é de cerca de dia embrionário 4 (E4). Aqui nós mostramos uma rápida e conveniente em técnica de eletroporação in ovo através do qual um DNA de plasmídeo que expressa a proteína fluorescente verde (GFP) como um marcador está diretamente entregue napinto espaço sub-retiniano embrionário e seguido por pulsos elétricos para facilitar a absorção de DNA por retina estaminais / progenitoras das células. O novo método de injecção de retina e electroporação em E4 permite a visualização de todos os tipos celulares da retina, incluindo a neurônios tardia-nascido 1, que tem sido difícil com o método convencional da injecção e electroporação em E1.5 2.

Watch this Scientific Journal Video about In Ovo Electroporation in Embryonic Chick Retina at JoVE.com

In Ovo Electroporation in Embryonic Chick Retina

O objetivo geral deste vídeo é mostrar como executar alvo injeção de retina e<em&gt; In ovo</em&gt; Electroporação de ADN / ARN construtos dentro da retina de pinto embrionário no Hamburger e do estágio Hamilton 22-23, o que é de cerca de dia embrionário 4 (E4). Esta técnica é muito útil para estudar a expressão do gene, regulação do gene, e mudança morfológica no desenvolvimento de retina do pintinho.

Em Eletroporação Ovo na retina de pinto embrionário

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is ...

in-ovo-electroporation-in-embryonic-chick-retina

Research

JoVE Journal

Biology

In Ovo Electroporation in Embryonic Chick Retina

14.3K visualizações.  University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School. O objetivo geral deste vídeo é mostrar como executar alvo injeção de retina e In ovo Electroporação de ADN / ARN construtos dentro da retina de pinto embrionário no Hamburger e do estágio Hamilton 22-23, o que é de cerca de dia embrionário 4 (E4). Esta técnica é muito útil para estudar a expressão do gene, regulação do gene, e mudança morfológica no desenvolvimento de retina do pintinho.

Vídeo: In Ovo Electroporation in Embryonic Chick Retina

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

Em Electroporations Ovo de Estágio HH 10 embriões de galinha

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

Biologia

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Método para coloração Whole Mount anticorpos em Pintainho

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

Ensaio para Indução Neural no embrião de galinha

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

Pintinho Ex ovo Cultura e Ex ovo CAM Ensaio: como ele realmente funciona

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Preparação de embriões de Microscopia Eletrônica do Drosophila Tubo cardíaco embrionário

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

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 ...

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

Decifrando Pathways Axonal dos Grupos geneticamente definidos de neurônios no tubo Neural Utilizando Pintainho In ovo Eletroporação

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Vídeo Bioinformática Análise de Crescimento Humano embrionárias Colony Stem Cell

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

Transfecção única célula em embriões de galinha

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

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 ...