Os autores gostariam de agradecer a Stephen Crews para dysfusion anticorpos, cDNA Dys e w voa 1118.

1. De colheita de embriões <ol><li> Configurar gaiolas a 25 ° C usando 2-4 dias de idade w placas de suco de 1118 flies.Grape são trocados a cada hora durante o dia para sincronizar a coleta de ovos ao longo de 1-2 período de dias antes da coleta </li><li> Coletar embriões para 1h a 25 ° C </li><li> Corte um pedaço retangular de ágar suco de uva, corte levemente no meio com uma lâmina de barbear, deixe uma linha no agar </li><li> Use uma sonda de metal para transferência de embriões a partir de chapa de suco de uva para o seu pedaço do ágar suco de uva, alinhá-los em uma linha reta com maior eixo do embrião em ângulo de 45 graus para a linha no meio do ágar. Obter uma linha reta de cerca de 50 embriões. Certifique-se de todos os pontos dos embriões na mesma direção, com extremidade posterior apontando para longe da linha. </li><li> Corte um pedaço retangular de fita dupla pau, e colocá-lo para uma lamínula 18x18mm. </li><li> Pick up embriões com fita dupla pau na lamínula </li><li> Coloque a lamínula para uma lâmina de vidro, secar o embrião no ar por cerca de 10 min. </li><li> Cobrir o embrião com uma fina camada de óleo de 700 Halocarbon, agora ele está pronto para a injecção. </li></ol> 2. Puxando Agulha Faça agulhas microinjeção puxando tubos capilares de vidro. Usamos um extrator de agulhas de Instrumento Mundial Precision (Modelo PUL-1). A agulha especificações do programa extrator são: calor: 1, demora 4. 3. Preenchimento das Agulhas Voltar carga de agulhas usando a pipeta Eppendorf p20 equipado com dicas Eppendorf Microloader, normalmente de carga 1,5-2 dsRNA mL 4. Quebrar o Needles <ol><li> Antes de quebrar a agulha, coloque uma lamínula sobre uma lâmina. </li><li> Prepare uma lâmina com uma gota de óleo no meio do slide </li><li> Ligue o III Picospritzer picopump para ajustar a pressão do ar </li><li> Quebrar a agulha cheia contra a borda de uma lamínula, criando uma ponta afiada. </li><li> Quando a ponta está quebrada, pisar no pedal de Picospritzer III picopump, alguns dsRNA vai vazar para fora da ponta da agulha. </li><li> Retire a lâmina com a lamínula. </li><li> Inserir a agulha sob a gota de óleo no meio da lâmina preparada antes. </li><li> Ajustar a definição sobre o III Picospritzer picopump para obter 100-200pl dsRNA quando você pisa no pedal. dsRNA pode ser visto como uma pequena gota. </li></ol> 5. Injeção <ol><li> Traga a ponta da agulha para a porção posterior do embrião blastoderma primeiro no mesmo plano focal e injetar o embrião fora do centro em torno de 30-50% de comprimento do ovo. </li><li> Depois de pisar no pedal do III Picospritzer picopump, uma pequena quantidade de dsRNA aparecerá como um pequeno ponto no local da injeção. </li></ol> 6. Análise fenotípica <ol><li> Após a injeção, coloque o slide em uma câmara úmida coberta (placa de Petri de plástico) a 18 ° C até que os embriões se desenvolvem ao estágio desejado embrionárias. </li><li> Use sonda para remover embriões a partir da fita dupla face e empurrá-los para a borda da lamínula. Lavar os embriões para fora do slide em um frasco de coleta com malha de nylon na parte inferior usando heptano, lavar 3 vezes com PBT. Embriões Dechorionate em lixívia 50% por 5 min, e lavar 3 vezes com PBT. Transferência de embriões a partir do tubo de coleta para um pedaço de malha de nylon, e depois mergulhar a tela de náilon na solução fixadora (heptano 5ml, 4,5 ml PBS, 0,5 ml de formol 37%) para liberar os embriões. Fix embriões na solução fixadora por 30min. Devitellinize embriões com metanol, transferência de embriões para um tubo eppendorf e immunostain os embriões utilizando protocolos padrão. </li><li> Embriões também podem desenvolver a fase larval apropriado. </li><li> Prepare uma lâmina com uma gota de óleo Halocarbon 700 no meio </li><li> Transferência de uma larva para o slide, coloque uma lamínula no topo </li><li> Observe larvas em microscópio de campo claro. </li></ol> 7. Resultados representativos: Um exemplo de dsRNA derrubar gene dysfusion (dis) em embrião Drosophlia é mostrado na Fig1. GFP-dsRNA embriões injetados são o controle negativo. Disfunção é um fator de transcrição bHLH PAS expressa em células de fusão traqueal. GFP-dsRNA embriões injetados mostrar a fusão da traquéia normal, e proteína Dys está presente em células de fusão (Figura 1A). Por outro lado, dis-dsRNA injetado embriões mostram fusão ramo falhou, e proteínas Dys não estão presentes em células de fusão (Figura 1B). Quando dsRNA injetado embriões se desenvolvem para a fase larval, as larvas dis-dsRNA injetado mostraram fusão ramo falhou (Fig.1D) em relação ao GFP-dsRNA injetado controle negativo (Figura 1C). Estes resultados mostraram eficazes derrubar de expressão de genes por disfunção endógena dsRNA injeção em embriões de Drosophila <img src="/files/ftp_upload/2477/2477fig1.jpg" alt="Figura 1" /> Figura 1. Remoção de função dis por dis-RNAi revela traqueal fusão defects. Embriões blastoderma (w 1118) foram injetados com qualquer GFP-dsRNA (controle negativo) ou dis-dsRNA e doseados por defeitos traqueal. (A) embrião Stage 16 injetados com GFP-dsRNA e corados com α-Dys e 2A12 mAb que as manchas do lúmen traqueal. Proeminentemente mostrado é o tronco lateral, que se fundiu. Setas apontam para as células tronco laterais Dys-positivos de fusão. (B) embrião Stage 16 injetados com dis-dsRNA e corados com α-Dys e 2A12 MAB. O embrião mostrou uma falta de fusão tronco lateral (o asterisco indica a localização normal de uma fusão-lateral do tronco em embriões de controle). Nenhuma célula Dys-positivos foram observados, indicando que a dis-RNA efetivamente aboliu a proteína Dys. (C a D) de larvas de segundo ínstar tanto com GFP-dsRNA ou dis-dsRNA foram examinadas por microscopia de campo brilhante para os defeitos da traquéia. (C) Sites de fusão são indicados por um asterisco na GFP-dsRNA larvas controle injetado. (D) O tronco laterais não fusível no larvas dis-RNA injetado, (*) indica a localização normal de uma fusão tronco lateral.

<ol>
	<li>St Johnston, D., Nusslein-Volhard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+pattern+and+polarity+in+the+Drosophila+embryo.">The origin of pattern and polarity in the Drosophila embryo.</a> Cell. 68, 201-219 (1992).</li><li>Venken, K. J., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+technologies+for+gene+manipulation+in+Drosophila+melanogaster.">Emerging technologies for gene manipulation in Drosophila melanogaster.</a> Nat. Rev. Genet. 6, 167-178 (2005).</li><li>Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans.">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391, 806-811 (1998).</li><li>Kennerdell, J. R., Carthew, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+dsRNA-mediated+genetic+interference+to+demonstrate+that+frizzled+and+frizzled+2+act+in+the+wingless+pathway.">Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway.</a> Cell. 95, 1017-1026 (1998).</li><li>Misquitta, L., Paterson, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+gene+function+in+Drosophila+by+RNA+interference+(RNA-i):+a+role+for+nautilus+in+embryonic+somatic+muscle+formation.">Targeted disruption of gene function in Drosophila by RNA interference (RNA-i): a role for nautilus in embryonic somatic muscle formation.</a> Proc. Natl. Acad. Sci. U. S. A. 96, 1451-1456 (1999).</li><li>Horowitz, A., Simons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Branching+morphogenesis.">Branching morphogenesis.</a> Circ. Res. 103, 784-795 (2008).</li><li>Hogan, B. L., Kolodziej, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organogenesis:+molecular+mechanisms+of+tubulogenesis.">Organogenesis: molecular mechanisms of tubulogenesis.</a> Nat. Rev. Genet. 3, 513-523 (2002).</li><li>Ghabrial, A., Luschnig, S., Metzstein, M. M., Krasnow, 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=Branching+morphogenesis+of+the+Drosophila+tracheal+system.">Branching morphogenesis of the Drosophila tracheal system.</a> Annu. Rev. Cell Dev. Biol. 19, 623-647 (2003).</li><li>Jiang, L., Crews, 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=Dysfusion+transcriptional+control+of+Drosophila+tracheal+migration,+adhesion,+and+fusion.">Dysfusion transcriptional control of Drosophila tracheal migration, adhesion, and fusion.</a> Mol. Cell. Biol. 26, 6547-6556 (2006).</li><li>Jiang, L., Crews, 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=The+Drosophila+dysfusion+basic+helix-loop-helix+(bHLH)-PAS+gene+controls+tracheal+fusion+and+levels+of+the+trachealess+bHLH-PAS+protein.">The Drosophila dysfusion basic helix-loop-helix (bHLH)-PAS gene controls tracheal fusion and levels of the trachealess bHLH-PAS protein.</a> Mol. Cell. Biol. 23, 5625-5637 (2003).</li></ol>

Não há conflitos de interesse declarados.

O dsRNA presentes método de injeção aqui permite uma análise muito sensível e rápido da função do gene em Drosophila desenvolvimento traqueal. Este método pode potencialmente ser aplicada para analisar a função do gene para outros tecidos e desenvolvimento do órgão.

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<td>Halocarbon oil 700</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>H8898</td>
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<td>Picospritzer III picopump</td>
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<td>Parker Precision Fluidics</td>
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rnai interference by dsrna injection into drosophila embryos

Screening genético é um dos métodos mais poderosos disponíveis para a obtenção de insights sobre processo biológico complexo 1. Ao longo dos anos muitas melhorias e ferramentas para a manipulação genética tornaram-se disponíveis em Drosophila 2. Logo após a descoberta inicial por Frie e Mello 3, que RNA de filamento duplo pode ser usado para knockdown a atividade de genes individuais em Caenorhabditis elegans, interferência de RNA (RNAi) foi mostrado para fornecer uma abordagem inversa genética poderosa para analisar funções de genes na Drosophila desenvolvimento dos órgãos 4, 5. Muitos órgãos, incluindo pulmões, rins, fígado e sistema vascular, são compostas de redes tubulares ramificados que os fluidos vitais de transporte ou gases 6, 7. A análise da formação de Drosophila traqueal fornece um sistema de excelente modelo para estudar a morfogênese de outros órgãos tubulares 8. O Berkeley Drosophila projeto genoma revelou centenas de genes que são expressos no sistema traqueal. Para estudar o mecanismo molecular e celular de formação do tubo, o desafio é compreender o papel destes genes no desenvolvimento traqueal. Aqui, descrevemos um método detalhado de dsRNA injeção em Drosophila embrião para knockdown expressão de genes individuais. Com sucesso derrubado endógena dysfusion expressão gênica (dis) por injeção dsRNA. Disfunção é uma proteína bHLH-PAS expressa em células de fusão de traquéia, e é necessária para a fusão ramo traqueal 9, 10. dis-RNAi completamente eliminada expressão disfunção e resultou em defeito de fusão traqueal. Este método relativamente simples fornece uma ferramenta para identificar genes requried para tissure e desenvolvimento de órgãos em Drosophila.

Watch this Scientific Journal Video about RNAi Interference by dsRNA Injection into Drosophila Embryos at JoVE.com

RNAi Interference by dsRNA Injection into Drosophila Embryos

Interferência de RNA tem se mostrado muito eficaz para analisar a função do gene em<em&gt; Drosophila</em&gt; Desenvolvimento traqueal. Um protocolo detalhado usada por Jiang laboratório para injetar dsRNA em embriões de voar para a expressão do gene knockdown é ilustrado. Esta técnica tem o potencial de genes de triagem necessária para o desenvolvimento de tecidos e órgãos em<em&gt; Drosophila</em&gt;.

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 ...

rnai-interference-by-dsrna-injection-into-drosophila-embryos

Nanyang Technological University

Research

JoVE Journal

Biology

RNAi Interference by dsRNA Injection into Drosophila Embryos

16.1K visualizações.  Oakland University. Interferência de RNA tem se mostrado muito eficaz para analisar a função do gene em Drosophila Desenvolvimento traqueal. Um protocolo detalhado usada por Jiang laboratório para injetar dsRNA em embriões de voar para a expressão do gene knockdown é ilustrado. Esta técnica tem o potencial de genes de triagem necessária para o desenvolvimento de tecidos e órgãos em Drosophila.

Vídeo: RNAi Interference by dsRNA Injection into Drosophila Embryos

Injection of dsRNA into Female A. aegypti Mosquitos

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus.  The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus. The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.

Injeção de dsRNA em Mosquitos Feminino A. aegypti

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

Biologia

Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This video protocol demonstrates a method used by the James laboratory to microinject DNA constructs into Anopheles stephensi embryos for the generation of transformed mosquitoes. Techniques for preparing microinjection needles, collecting and preparing embryos and performing the microinjection are illustrated.

Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.

Uma injeção de. Embriões stephensi para gerar malária resistente a mosquitos

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This ...

In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos

In-utero in-vivo injection and electroporation of the embryonic rat 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 rats 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 E15-E21 rats, however it is most commonly performed at E16 as shown in this video.

In Utero injeção intraventricular e eletroporação de embriões de ratos E16

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

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

Dissecação ao vivo de Drosophila Embriões: Métodos ágeis para Screening Coleções Mutant pela coloração de anticorpos

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

In vivo Centrifugação de Embriões Drosophila

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

Entrega bacteriana de RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

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, ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

Imagem vertical de Drosophila Os embriões

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Culturas de células primárias de Drosophila Os embriões Gástrula

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

Imagem Alterar Forma Celular em Viver Drosophila Os embriões

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...