Este protocolo é usado atualmente em nosso laboratório e tem sido aperfeiçoado ao longo dos anos por muitas pessoas, incluindo os Drs. David Sharp, Kwon Mijung, Sommi Patrizia, Cheerambathur dhanya. Agradecemos a Dr Bill Sullivan (UCSC), que nos forneceu excelentes conselhos sobre a manipulação e microinjeção de embriões Drosophila quando o nosso trabalho neste sistema estava sendo iniciada (ref. 13). Agradecemos a todos os membros do laboratório Scholey. Nosso trabalho sobre mitose em Drosophila é suportado pelo NIH conceder GM55507.

Receitas: Suco de uva placas: <ul><li> 5,5 g de ágar Bacto </li><li> 14,5 g de dextrose ou glicose </li><li> 7,15 g de sacarose </li><li> 45 ml suco concentrado de uva (100% suco). </li><li> 204,5 ml H 2 0 </li><li> 625 mL de NaOH 10N </li></ul> Misture todos os ingredientes e microondas até ferver. Adicionar 2,8 ml de ácido mix (mistura de ácido: 20,9 ml de ácido propiônico, ácido fosfórico 2,1 ml, 27 ml H 2 0) Misture e despeje em 35 milímetros pratos de Petri. Deixe solidificar à temperatura ambiente por um ou dois dias. Se as placas não vão ser usados ​​em breve, vedação com parafilme e manter a 4 ° C (deixar equilibrar à temperatura ambiente antes de usar). Colar de levedura: Dissolva cerca de uma colher de chá de fermento (Sigma YSC2, levedura de Saccharomyces cerevisiae tipo II) em água para formar uma pasta grossa. Coloque uma pequena quantidade deste em cada prato suco de uva pouco antes de usar. G-PEM buffer: <ul><li> 80 mM Na-PIPES, pH 6,9 </li><li> 1 mM MgCl 2 </li><li> 1 mM EGTA </li><li> 1 mM GTP </li></ul> Buffer de injecção: <ul><li> 150 mM K-Aspartato </li><li> 10 mM K-fosfato </li><li> Imidazol 20 mM, pH 7,2 </li></ul> Cola heptano: Desenrole fita dupla adesiva e coloque em uma garrafa de 100ml, adicionar cerca de 50ml heptano, selar a garrafa, e rock durante vários dias. Ao usar, adicione heptano se a cola é muito grosso. Câmara de desidratação: Dê uma placa de Petri 100 milímetros, coloque uma parte de um prato 35 milímetros para dentro para fazer uma &quot;mesa&quot; e adicione Drierite (sulfato de cálcio anidro) em torno dele para que a altura de Drierite não é maior do que a &quot;mesa&quot; e cobertura. A lamela com embriões serão colocados nesta &quot;tabela&quot; para a desidratação antes da injeção. É uma boa idéia para usar pelo menos alguns Drierite indicando e alterá-lo quando ele mudou de cor. Protocolo: Este protocolo pode ser utilizado para a injeção de praticamente qualquer reagente solúvel no embrião Drosophila sincicial: Por exemplo, seja uma proteína fluorescente para observação ou um inibidor da proteína-alvo, ou ambos. Ficando tudo pronto: 1 .- 2 a 3 dias antes do experimento, fazer placas de suco de uva e configurar leigos gaiola: Pegue uma garrafa de plástico (6 oz, fundo redondo de Drosophila Científica Aplicada), corte dois pequenos furos (cerca de 1cm 2) em lados opostos do garrafa e preencha com um pedaço de algodão (o que permitirá que o fluxo de ar), toque as moscas dentro da garrafa e cubra com uma placa de suco de uva. Mantenha a 25 ° C (ou à temperatura ambiente), as moscas devem estabelecer embriões para cerca de 10 dias. 2 .- Pelo menos um dia antes do experimento puxar agulhas para injeção, usamos uma agulha Kopf / Pipetar modelo extrator 720 e finos filamentos de vidro paredes (Precision Instruments tw100f Mundial). Para injeção de tubulina fluorescentes, manter agulhas a 4 ° C para evitar induzidas pela temperatura de polimerização na agulha. Preparação tubulina: Nota: Todos os trabalhos com tubulina deve ser feito a 4 ° C para evitar a polimerização. <ol><li> Tome tubulina rotulados fora do congelador e colocá-lo em gelo durante 5 a 15 minutos. </li><li> Diluir com G-PEM tampão (4 a 10 vezes, dependendo da intensidade necessária). </li><li> Centrifugar a 13k rpm a 4 ° C por 10-15 minutos. </li><li> De carga 1 - 2 l na agulha. </li></ol> Anticorpo ou inibidor químico: Prepare agulhas como para tubulina, usando uma concentração adequada (isto pode precisar de ser testado). O anticorpo pode ser em tampão de injeção, G-PEM buffer ou PBS. Se outro buffer é necessária, esse buffer terá de ser injetado como um controle para garantir que este tampão não causa nenhum dano por conta própria. Nota: tubulina Inibidor e fluorescentes podem ser misturados e injectados em conjunto, se as concentrações permitem. Caso contrário, injetar o tubulina fluorescentes e esperar 5 a 10 minutos antes de injetar o inibidor. Ao realizar uma injeção dupla, comece com mais embriões, uma vez que menos embriões irá recuperar normalmente. De colheita de embriões: <ol><li> Colocar uma placa de suco de uva novo na gaiola leigos. </li><li> Remover esta placa, após uma hora, esta é a primeira coleção do dia e muitas vezes não é muito bom. Ele pode ser descartado. </li><li> Mudar a placa a cada hora para manter embriões coleta de uma hora cada. </li></ol> Preparação lamela: <ol><li> Coloque uma lamela 50 x 22 milímetros em um lado de uma lâmina de microscópio. Tape os quatro cantos para o slide para que ele não se move. </li><li> Usando um aplicador derrubado algodão, coloque uma camada de cola heptano em uma única linha sobre a lamela (a cola não deve ser viscoso e deve secar em poucos segundos, caso contrário, adicionar mais heptano). </li><li> Coloque um pedaço dedupla fita adesiva sobre o slide para o lado da lamela. </li></ol> Preparação embrião: Nota: Os embriões devem ser visualizados cerca de 2 horas após o início da coleta, para começar os seguintes passos permitindo tempo suficiente para terminá-los dentro desse espaço de tempo. <ol><li> Com um pincel umedecido com cuidado pegar os embriões da placa de suco de uva e coloque em fita adesiva dupla no slide. </li><li> Usando a parte exterior das pinças, rolar os embriões sobre a fita adesiva dupla até o córion (membrana externa) se rompe. </li><li> Pegue o embrião de suavemente ondulado-lo sobre o córion assim que adere à pinça e colocá-lo na cola heptano na lamela com o lado longo do paralelo de embriões para o lado longo da lamela. Conjunto de 10 a 20 embriões em uma linha. </li><li> Retirar as lamelas e colocá-lo na câmara de desidratação de 3-8 minutos (isso depende da umidade da sala ea quantidade a ser injetada). </li><li> Coloque em uma câmara metálica com graxa de vácuo. </li><li> Cobrir os embriões com 700 halocarbono óleo para evitar uma maior desidratação. Embriões já estão prontos para injeção. </li></ol> Injeção de embrião: <ol><li> Pesquisar os embriões em um objetivo 16x. </li><li> Mover os embriões de distância, e encontrar a agulha sem mover o plano focal. </li><li> Centro de agulha e movê-lo sem movê-lo na direção x ou y. </li><li> Se a agulha não estiver aberto, colocar a ponta da lamela no campo de visão, mas não onde a agulha será, e inferior a agulha para o mesmo plano focal. Com muito cuidado mover a lamela até atingir a agulha e delicadamente quebra-la aberta. Se a agulha estiver aberto, vá para o passo 5. (As agulhas podem ser abertos com ácido fluorídrico antes de preencher). </li><li> Colocar os embriões em ver (mas não onde a agulha será), menor a agulha no óleo e certifique-se de obter gotas de líquido agradável da agulha. </li><li> Com cuidado mas firmemente mover o embrião para a agulha e injetar uma gota no embrião e mover o embrião de distância. (Ou siga as instruções do seu aparelho de injecção). </li><li> Depois de todos os embriões foram injetados, eles estão prontos para observação em um microscópio confocal. </li></ol> Imagem: Imagem os embriões em um microscópio confocal com um objetivo de 60 ou 100 vezes: Notas: <ol><li> O intervalo de tempo em uma série de lapso de tempo deve ser no máximo alguns segundos, uma vez que a mitose no embrião é muito rápido, dependendo do processo exato a ser estudado. </li><li> Uma série 3D ou um único plano pode ser adquirido, dependendo do processo de interesse. </li><li> Para os inibidores de ação rápida, a transferência do microscópio de injeção para microscópio de imagem tem que ser rápido. </li></ol><img src="/files/ftp_upload/1382/1382fig1.jpg" alt="figura 1" /> Figura 1: Estudo da mitose no embrião Drosophila sincicial. Núcleos no embrião sofrer divisões rápida sem citocinese, durante os ciclos de 10 a 13 núcleos formam uma monocamada no córtex. A. Esquemática do embrião com núcleos no córtex. Embrião pode expressar GFP e / ou RFP proteínas e pode ser rotulada microinjeção com outras proteínas rotuladas e / ou inibidores. B. imagem lapso de tempo de embrião expressando GFP-tubulina e RFP-histonas. Plot C. do pólo-pólo de separação para ciclos de 11 , 12 e 13 em um embrião de tipo selvagem. Comprimento do eixo apresenta períodos de duração isométrica e períodos de alongamento, que são muito consistentes e reprodutíveis. Dynamics D. dos microtúbulos do fuso. Injeção de baixa concentração de tubulina rodamina leva a manchas formadas por subunidades de tubulina poucos, o que pode ser usado para estudar a dinâmica de microtúbulos.

<ol>
	<li>Brust-Mascher, I., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+spindle+dynamics+in+Drosophila.">Mitotic spindle dynamics in Drosophila.</a> Int Rev Cytol. 259, 139-172 (2007).</li><li>Duffy, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GAL4+system+in+Drosophila:+a+fly+geneticist's+Swiss+army+knife.">GAL4 system in Drosophila: a fly geneticist's Swiss army knife.</a> Genesis. 34, 1-15 (2002).</li><li>Morris, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+methods+for+analyzing+the+functions+of+kinesins+in+early+embryos.">Microinjection methods for analyzing the functions of kinesins in early embryos.</a> Methods Mol Biol. 164, 163-172 (2001).</li><li>Brust-Mascher, I., Sommi, P., Cheerambathur, D. K., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinesin-5-dependent+poleward+flux+and+spindle+length+control+in+Drosophila+embryo+mitosis.">Kinesin-5-dependent poleward flux and spindle length control in Drosophila embryo mitosis.</a> Mol Biol Cell. 20, 1749-1762 (2009).</li><li>Brust-Mascher, I., Civelekoglu-Scholey, G., Kwon, M., Mogilner, A., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+for+anaphase+B:+role+of+three+mitotic+motors+in+a+switch+from+poleward+flux+to+spindle+elongation.">Model for anaphase B: role of three mitotic motors in a switch from poleward flux to spindle elongation.</a> Proc Natl Acad Sci U S A. 101, 15938-15943 (2004).</li><li>Brust-Mascher, I., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+flux+and+sliding+in+mitotic+spindles+of+Drosophila+embryos.">Microtubule flux and sliding in mitotic spindles of Drosophila embryos.</a> Mol Biol Cell. 13, 3967-3975 (2002).</li><li>Cheerambathur, D. K., Brust-Mascher, I., Civelekoglu-Scholey, G., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+partitioning+of+mitotic+kinesin-5+crosslinkers+between+microtubule-bound+and+freely+diffusing+states.">Dynamic partitioning of mitotic kinesin-5 crosslinkers between microtubule-bound and freely diffusing states.</a> J Cell Biol. 182, 421-428 (2008).</li><li>Cheerambathur, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+an+anaphase+B+switch:+predicted+role+for+a+microtubule+catastrophe+gradient.">Quantitative analysis of an anaphase B switch: predicted role for a microtubule catastrophe gradient.</a> J Cell Biol. 177, 995-1004 (2007).</li><li>Kwon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chromokinesin,+KLP3A,+dives+mitotic+spindle+pole+separation+during+prometaphase+and+anaphase+and+facilitates+chromatid+motility.">The chromokinesin, KLP3A, dives mitotic spindle pole separation during prometaphase and anaphase and facilitates chromatid motility.</a> Mol Biol Cell. 15, 219-233 (2004).</li><li>Sharp, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+coordination+of+three+mitotic+motors+in+Drosophila+embryos.">Functional coordination of three mitotic motors in Drosophila embryos.</a> Mol Biol Cell. 11, 241-253 (2000).</li><li>Sharp, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bipolar+kinesin,+KLP61F,+cross-links+microtubules+within+interpolar+microtubule+bundles+of+Drosophila+embryonic+mitotic+spindles.">The bipolar kinesin, KLP61F, cross-links microtubules within interpolar microtubule bundles of Drosophila embryonic mitotic spindles.</a> J Cell Biol. 144, 125-138 (1999).</li><li>Sharp, D. J., Rogers, G. C., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+dynein+is+required+for+poleward+chromosome+movement+during+mitosis+in+Drosophila+embryos.">Cytoplasmic dynein is required for poleward chromosome movement during mitosis in Drosophila embryos.</a> Nat Cell Biol. 2, 922-930 (2000).</li><li>Sharp, D. J., Yu, K. R., Sisson, J. C., Sullivan, W., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antagonistic+microtubule-sliding+motors+position+mitotic+centrosomes+in+Drosophila+early+embryos.">Antagonistic microtubule-sliding motors position mitotic centrosomes in Drosophila early embryos.</a> Nat Cell Biol. 1, 51-54 (1999).</li><li>Waterman-Storer, C. M., Desai, A., Bulinski, J. C., Salmon, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+speckle+microscopy,+a+method+to+visualize+the+dynamics+of+protein+assemblies+in+living+cells.">Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells.</a> Curr Biol. 8, 1227-1230 (1998).</li></ol>

Este protocolo é relativamente simples, no entanto, cada etapa requer prática para se certificar de que os embriões não estão danificados. Experimentos de controle cuidadoso sempre precisa ser feito para garantir que os resultados estão sendo obtidos confiável. Uma boa maneira de começar isso é microinjecting tubulina buffer ou rodamina em embriões de controle expressando GFP-tubulina para se certificar de que a mitose prossegue normalmente através de todos os ciclos na superfície do embrião (ciclos de 10 a...

microinjection techniques for studying mitosis in the drosophila melanogaster syncytial embryo

Este protocolo descreve o uso do embrião Drosophila melanogaster sincicial para estudar a mitose 1. Drosophila tem uma genética útil com um genoma sequenciado, e pode ser facilmente mantido e manipulado. Mutantes mitose existem muitas, e as moscas transgênicas expressando funcional fluorescente (GFP, por exemplo) - proteínas mitótica tagged foram e estão sendo gerados. Expressão do gene alvo é possível utilizar o sistema GAL4/UAS 2. O embrião Drosophila cedo realiza múltiplas mitoses muito rapidamente (duração do ciclo celular, ≈ 10 min). É bem adequado para imagens de mitose, pois durante os ciclos de 10-13, os núcleos se dividem rapidamente e de forma síncrona sem intervir citocinese na superfície do embrião em uma monocamada única logo abaixo do córtex. Esses núcleos se dividem rapidamente, provavelmente, usar o aparelho mitótico mesmo que outras células, mas eles são otimizados para a velocidade, o ponto de verificação é acreditado geralmente para não ser rigoroso, permitindo o estudo de proteínas mitótica cuja ausência poderia causar parada do ciclo celular em células com um posto de controle forte . Embriões que expressam proteínas GFP rotulados quanto com microinjeção de proteínas fluorescentes rotulados podem ser facilmente visualizados para acompanhar a dinâmica ao vivo (Fig. 1). Além disso, os embriões podem ser microinjeção com função de bloqueio de anticorpos ou inibidores de proteínas específicas para estudar o efeito da perda ou perturbação da sua função 3. Estes reagentes podem se propagar por todo o embrião, atingindo muitos fusos para produzir um gradiente de concentração de inibidor, o que resulta em um gradiente de defeitos comparável a uma série alélica de mutantes. Idealmente, se a proteína-alvo é fluorescente etiquetado, o gradiente de inibição pode ser diretamente visualizada 4. Supõe-se que o mais forte fenótipo é comparável ao fenótipo nulo, embora seja difícil formalmente excluir a possibilidade de que os anticorpos podem ter efeitos dominante em casos raros, os controles de modo rigoroso e interpretação cautelosa deve ser aplicado. Mais longe do local da injeção, a função da proteína é apenas parcialmente perdido permitindo que outras funções da proteína-alvo para se tornar evidente.

Watch this Scientific Journal Video about Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo at JoVE.com

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

Este protocolo descreve o uso de microinjeção e imagens de alta resolução na<em&gt; Drosophila melanogaster</em&gt; Sincicial embrião para estudar a mitose.

Técnicas de microinjeção para estudar na Mitose Drosophila melanogaster Embrião Sincicial

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

microinjection-techniques-for-studying-mitosis-drosophila

Research

JoVE Journal

Biology

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

14.2K visualizações.  University of California, Davis. Este protocolo descreve o uso de microinjeção e imagens de alta resolução na Drosophila melanogaster Sincicial embrião para estudar a mitose.

Vídeo: Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

Dissecção do CNS Larval em Drosophila melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Biologia

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

De alta resolução de vídeo de Rastreamento Locomotion em Adult Drosophila melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

Gravação eletrofisiológicas na Drosophila Embrião

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

Função de monitorização cardíaca em larval Drosophila melanogaster Para estudos fisiológicos

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

Grampo patch e Técnicas de Perfusão para estudar canais iônicos Expresso em Xenopus Oócitos

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

Estudar checkpoint mitótico por Ilustrando comportamento dinâmico cinetocoro Proteína e Movimento de cromossomos em Viver<em&gt; Drosophila</em&gt; Os embriões Sincicial

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

Medição do Tempo de vida em<em&gt; Drosophila melanogaster</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

Imagem Através do Pupal Caso de<em&gt; Drosophila melanogaster</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular biology has been greatly fueled by the discovery of noncoding RNAs as major players in post-transcriptional regulation. Such a revolutionary perspective has been accompanied and triggered by the development of powerful technologies for profiling short RNAs expression, both at the high-throughput level (genome-wide identification) or as single-candidate analysis (steady state accumulation of specific species). Although several state-of-art strategies are currently available for dosing or visualizing such fleeing molecules, Northern Blot assay remains the eligible approach in molecular biology for immediate and accurate evaluation of RNA expression. It represents a first step toward the application of more sophisticated, costly technologies and, in many cases, remains a preferential method to easily gain insights into RNA biology. Here we overview an efficient protocol (Enhanced Northern Blot) for detecting weakly expressed microRNAs (or other small regulatory RNA species) from Drosophila melanogaster whole embryos, manually dissected larval/adult tissues or in vitro cultured cells. A very limited amount of RNA is required and the use of material from flow cytometry-isolated cells can be also envisaged.

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.

Aprimorada do Norte Detecção Blot de pequeno RNA de espécies em<em&gt; Drosophila melanogaster</em

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.

Técnicas de microinjeção para estudar na Mitose Drosophila melanogaster Embrião Sincicial

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