Este trabalho foi suportado por concessões para AK da Ciência Israel Foundation, Israel O ministério da saúde, e DFG (Fundação Alemã de Pesquisa).

I. Eletroporação Manipulação de ovos <ol><li> Coloque os ovos em um conjunto de incubadora umidificada a 37-38 ° C, de preferência uma incubadora com um bandejas de balanço. </li><li> Embrião são electroporated após cerca de 66 horas de incubação, quando atingiram Hamburger &amp; Hamilton estágio (HH) 18-20. Nesta fase, a cabeça encontra-se em ângulos retos com o tronco (condição chamada de flexão cervical) e um vasto conjunto de vasos extra-embrionárias do sangue, como o anterior, posterior, direita e esquerda veias vitelina deve ser visto. Esta é uma fase ótima para enhancer-dependente expressão específica 1,3,4. É importante monitorar a temperatura e umidade da incubadora, a fim de obter embriões viáveis ​​e sincronizados. </li><li> Após 66 horas de incubação, retirar os ovos da incubadora. Coloque os ovos na horizontal. Espere 5-10 minutos. O embrião agora está localizada no pólo superior do ovo. </li></ol> Preparativos <ol><li> Preparar a solução de Hank com penicilina estreptomicina / (P / S) (1:100). </li><li> Puxe capilares de vidro (0,5 mm de diâmetro) para microcapilar. </li><li> Coloque os eletrodos (tungstênio) em micromanipulador e conectar eletrodos ao gerador de pulsos (ECM 830). </li><li> Ajustar o gerador de pulsos com os seguintes parâmetros: 30V de tensão, número de pulsos - 3, comprimento do pulso de 50 ms. </li><li> Prepare uma pipeta na boca, uma agulha de seringa e uma fita. </li><li> Prepare uma mistura de DNA desejado (2-5μg/μl) e adicionar um corante Fast Green. </li></ol> Janelas e eletroporação <ol><li> Usando uma seringa, remova 5-6 ml de albumina de cada ovo. </li><li> Com uma tesoura pequena e aberta uma janela oval na parte superior do ovo. </li><li> Embrião úmido com 0,5-1ml de Hank + solução de P / S. </li><li> Use uma pipeta de boca, para carregar o vidro microcapilar com a mistura de DNA. </li><li> Coloque o embrião com sua cauda em sua direção. Nesta fase, a medula espinhal de embriões é claramente visto. Assim, não técnicas de contraste são necessários. A medula espinhal é selado em ambas as extremidades, a cabeça ea cauda, ​​mas se o seu microcapilar é fina o suficiente, você será capaz de furar um buraco pequeno e penetrar no tubo neural em um ângulo raso. A microcapilar de diâmetro direito deve ser carregado facilmente com DNA, penetrar no tubo sem danificá-lo e liberar o DNA com uma exalação regular. </li><li> Injetar o DNA com uma pipeta de boca. O corante verde deve se espalhar a partir da ponta da cauda até as vesículas do cérebro em desenvolvimento. </li><li> Colocar os eletrodos em paralelo ao tubo neural, tão perto quanto você pode, sem tocar o próprio tubo e pulso. No momento do pulso, os eletrodos devem ser cobertos com o líquido para permitir condutividade elétrica. Normalmente, a quantidade de Hank é adicionado na mendicidade é suficiente. Se não adicionar uma gota de apenas de Hank antes do pulso. </li><li> Remova cuidadosamente os eletrodos e selar a janela com uma fita. É importante para selar o ovo inteiramente para evitar a secagem do embrião durante o período de incubação seguinte. </li><li> Coloque o ovo de volta na incubadora. </li></ol> II. Preparação medula espinhal livro aberto Descolamento da medula espinhal do embrião <ol><li> Remover o embrião eletroporated, em E6, do ovo e coloque-o em um prato de Petri revestidas com silicone contendo PBS. </li><li> Cortar as membranas e esticar o embrião do seu lado ventral utilizando pinos para prendê-lo. </li><li> Usando um tungstênio afiadas microcapilar fazer uma incisão longitudinal ao longo da placa do telhado, do rombencéfalo até a cauda. </li><li> Faça um adicional de duas incisões longitudinais em ambos os lados da medula espinhal, destacando-se os gânglios da raiz dorsal (DRGs) de distância da medula espinhal. </li><li> Destacar a placa de fundo a partir do tecido a partir da cauda para o rombencéfalo, deixando a medula espinhal intacta. </li><li> Corte da medula espinhal em uma seção transversal na parte posterior do cérebro usando uma tesoura fina e separá-lo do corpo. </li></ol> Fixação <ol><li> Espalhe o cordão espinhal isolado numa placa de Petri novo revestido com silicone contendo PBS, utilizando os pinos para prendê-lo, a partir do cérebro posterior à cauda produzir uma preparação flat-mount. </li><li> Despeje o PBS do prato e substituí-lo com paraformaldeído 4% em tampão fosfato. </li><li> Incubar a temperatura ambiente por 1 hora. </li></ol> Imuno-histoquímica <ol><li> Transferir o cabo fixo da coluna vertebral em um frasco contendo anticorpo primário em PBS. </li><li> Incube com agitações suave durante a noite a 4 ° C. </li><li> Lavar o anticorpo X5 vezes com PBS, cada lavagem durante 1 hora com agitação suave. </li><li> Adicionar anticorpo secundário e incubar com agitações suave durante a noite a 4 ° C. </li><li> Lavar o anticorpo X5 vezes com PBS, cadalavar por 1 hora com agitação suave. </li></ol> Montagem de livro aberto para imagens <ol><li> Esfregaço graxa ou silicone em forma de retângulo sobre uma lâmina e gotejar PBS dentro. </li><li> Coloque a medula espinhal esticada no meio do retângulo, usando uma pinça e extrair o líquido em torno dele com uma pipeta Pasteur. </li><li> Coloque 1-2 gotas de meios de montagem sobre a medula espinhal e cobrir com lamínula. Esmagá-lo para evitar bolhas de ar. </li><li> Manter os slides no escuro em 4 ° C. </li><li> Inspecionar a medula espinhal através de microscopia confocal. </li></ol>

<ol>
	<li>Zisman, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolysis+and+membrane+capture+of+F-spondin+generates+combinatorial+guidance+cues+from+a+single+molecule.">Proteolysis and membrane capture of F-spondin generates combinatorial guidance cues from a single molecule.</a> J Cell Biol. 178 (7), 1237-1249 (2007).</li><li>Arakawa, T., Iwashita, M., Matsuzaki, F., Suzuki, T., Yamamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paths,+elongation,+and+projections+of+ascending+chick+embryonic+spinal+commissural+neurons+after+crossing+the+floor+plate.">Paths, elongation, and projections of ascending chick embryonic spinal commissural neurons after crossing the floor plate.</a> Brain Res. 1223, 25-33 (2008).</li><li>Avraham, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+control+of+axonal+guidance+and+sorting+in+dorsal+interneurons+by+the+Lim-HD+proteins+Lhx9+and+Lhx1.">Transcriptional control of axonal guidance and sorting in dorsal interneurons by the Lim-HD proteins Lhx9 and Lhx1.</a> Neural Dev. 4 (1), 21 (2009).</li><li>Timmer, J., Johnson, J., Niswander, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+in+ovo+electroporation+for+the+rapid+analysis+of+neural-specific+murine+enhancers.">The use of in ovo electroporation for the rapid analysis of neural-specific murine enhancers.</a> Genesis. 29 (3), 123-132 (2001).</li><li>Reeber, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulating+Robo+expression+in+vivo+perturbs+commissural+axon+pathfinding+in+the+chick+spinal+cord.">Manipulating Robo expression in vivo perturbs commissural axon pathfinding in the chick spinal cord.</a> J Neurosci. 28 (35), 8698-8708 (2008).</li></ol>

Eletroporação de DNA plasmídeo no embrião de galinha evolui como uma poderosa técnica para na expressão ectópica vivo. A combinação de enhancers específico, e eletroporação pinto fornece uma ferramenta rápida e eficiente para decifrar caminhos axonal de um grupo geneticamente definido de neurônios 1,3,5. Utilizando o Cre / LoxP e os sistemas de amplificação Gal4/UAS pode aumentar a intensidade e duração da expressão. A imagem que surge é de uma divergência complexo de pistas axonal que su...

deciphering axonal pathways of genetically defined groups of neurons in the chick neural tube utilizing in ovo electroporation

Emprego de elementos enhancer para dirigir a expressão de genes repórter em neurônios é um paradigma amplamente utilizado para rastreamento de projeção axonal. Para rastreamento de projeção axonal de interneurônios da coluna vertebral nos vertebrados, os genes de células germinativas alvo repórter rendimento rotulagem bilateralmente simétrica. Portanto, é difícil distinguir entre o ipsi-e contra-lateralmente axônios de projeção. Eletroporação unilateral dentro do tubo neural pinto fornece um meio útil para restringir a expressão de um gene repórter para um lado do sistema nervoso central, e seguir a projeção axonal em ambos os lados<sup&gt; 1 ,2-5</sup&gt;. Este vídeo demonstra primeiro como lidar com os ovos antes da injeção. No estágio HH 18-20, DNA é injetado no nível sacral do tubo neural, então, eletrodos de tungstênio são colocados em paralelo ao embrião e curta pulsos elétricos são administrados com um gerador de pulsos. O ovo é selada com fita adesiva e colocados de volta para uma incubadora para o desenvolvimento. Três dias depois (E6) a medula espinhal é removido como uma preparação de livro aberto de embrião, fixa, e processados ​​para a coloração de toda anticorpo de montagem. O cordão espinhal manchada é montado na corrediça e visualizados através de microscopia confocal.

Watch this Scientific Journal Video about Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation at JoVE.com

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

Este vídeo demonstra como visualizar caminhos axonal de grupos geneticamente definido de neurônios na medula espinhal utilizando pinto embrionárias<em&gt; In ovo</em&gt; Eletroporação de genes repórter sob o controle de elementos específicos enhancer.

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 ...

deciphering-axonal-pathways-genetically-defined-groups-neurons-chick

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

9.2K visualizações.  Institute for Medical Research Israel Canada, Hebrew University-Hadassah Medical School. Este vídeo demonstra como visualizar caminhos axonal de grupos geneticamente definido de neurônios na medula espinhal utilizando pinto embrionárias In ovo Eletroporação de genes repórter sob o controle de elementos específicos enhancer.

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

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

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 ...

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

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

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 ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purificação do M. magneticum Strain AMB-1 proteína associada Magnetosome MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

Rastreamento Deformações Tissue Morfogenéticas no embrião de galinha precoce

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

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 Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons1, which has been difficult with the conventional method of injection and electroporation at E1.52.

In Ovo Electroporation in Embryonic Chick Retina

The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.

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 ...