Este trabalho foi suportado por uma concessão do National Science Foundation (MCB-0923794 para FT).

1. Produzindo peritécios <ol><li> Centro de ágar cenoura inocular 10 em um centímetro de diâmetro 6,0 prato de Petri com uma cepa fértil de F. graminearum (Recomenda PH-1). </li><li> O local de inoculação de pratos sob brilhantes luzes fluorescentes à temperatura ambiente (18-24 ° C) e deixa-se crescer até micélio tenha atingido o bordo exterior da placa de Petri (3-5 dias). Luzes fluorescentes domésticas comerciais funcionam bem para estimular a formação de frutificação corpo e descarga. </li><li> Remova cuidadosamente o micélio aéreo com um palito estéril. </li><li> Distribuir 1,0 ml 2,5% de Tween 60 aq à superfície com uma hockeystick vidro estéril ou extremidade arredondada de uma vareta de vidro estéril. </li><li> Retornar placas à luz. Não adicione Parafilm para placas. </li><li> Após 24 h, a superfície das placas devem ter uma aparência brilhante. Se micélios reaparece, raspagem de superfície e reaplicar Tween-60 solução. </li><li> Observe o desenvolvimento de peritécios na próxima semana. Os estágios intermediários dedesenvolvimento peritécio pode ser colhido por suavemente raspagem da superfície do agar e flash congelado para RNA de extracção (Figura 1). </li><li> Esporos maduros irá acumular-se na tampa do prato por dia 7. Inicialmente, estes só será visível sob um microscópio de dissecação, mas por dia 9, eles irão ter acumulado à densidade suficiente de modo a ser visível a olho nu. </li></ol> 2. Ensaio de descarga de ascósporos <ol><li> No dia 6 após a aplicação da solução de Tween, cortar um círculo 1 cm de diâmetro para fora do ágar com uma broca de madeira. Alternativamente, um bisturi pode ser usado para remover um segmento de dimensões semelhantes. O círculo pode ser cortado ao meio e cada metade colocada numa lâmina de microscópio de vidro. </li><li> Orientar as peças para a superfície contendo os corpos de frutificação é perpendicular à superfície da lâmina, e os esporos são disparados ao longo do comprimento da lâmina. </li><li> Colocar a lâmina em uma câmara de umidade durante a noite sob as luzes. Esporos irá acumular sobrea corrediça e ser visível a olho nu na manhã seguinte (Figura 2). </li><li> Esporos acumulados podem ser lavadas para fora do slide com água e quantificados, se desejado. </li><li> Potenciais inibidores de descarga de ascósporos pode ser ensaiada adicionando-os para o lado de trás do bloco de agar durante a montagem do ensaio. Alguns inibidores de canais iónicos que reduzem descarga foram previamente testados com esta técnica 15. </li></ol> 3. Geração de progênie recombinante <ol><li> Iniciado atravessar através da inoculação em ágar cenoura com duas cepas de F. graminearum (um Nit + e um nit-), colocando os pontos de inoculação em lados opostos da placa de Petri. </li><li> Uma vez que as hifas ter enchido a superfície, raspar parte aérea e aplicar Tween-60 solução como acima descrito. Use um marcador notar, no lado da placa de Petri onde as hifas se encontram. </li><li> Voltar culturas à luz e incubar até peritécios têm desenvolvido, e ter barbilhoes serarma para formar a partir do peritécio ao longo da intersecção entre as duas culturas. </li><li> Sob o microscópio de dissecação, pôr em ver a peritécios ao longo da intersecção das duas culturas. Usando um pino de insectos, esterilizados e mergulhado em água estéril, um toque cirrhus ao longo desta fronteira levemente com a ponta do pino. A umidade no pino deve permitir que os cirrhus para manter o pino. </li><li> Lavar a extremidade do pino com os cirrhus sobre ele em 200 uL de água estéril num tubo de Eppendorf para lavar os esporos fora. </li><li> Chama a pino, umedecê-la novamente e escolher outra cirrhus. Escolha 10-15 barbilhoes, e colocar cada em um tubo separado de água. </li><li> Resumidamente tubos vortex contendo suspenso barbilhoes. </li><li> Remover 60 uL da suspensão de esporos com uma pipeta, coloque em MMTS meio 1 em um prato 9 centímetros de Petri. Espalhe com um taco de hóquei estéril. A suspensão de esporos restante pode ser armazenado a 4 ° C durante até uma semana e replated se necessário. </li><li> Incubaçãote à temperatura ambiente (luz não necessário) durante 3-5 dias até as colónias muito pequenas aparecem (Figura 3). Haverá dois tipos de fenótipos entre as colônias. As colônias de nit terá hifas esparsa que as colónias + NIT. Barbilhoes de recombinante peritécios irá mostrar ambos os fenótipos de colônias em proporção aproximadamente igual. Descartar as placas com colônias de apenas um único fenótipo sobre eles. Note que às vezes mais de dois fenótipos resultar devido à segregação de outros fatores. </li><li> Mova colônias de V8 ou ágar cenoura para o armazenamento. Colónias de teste em Agar Czapek Dox (os estirpes nit-não vai crescer em Dox Czapek) e em PDA com clorato (0,5 M clorato de potássio; as estirpes + de nit não vai crescer em PDA com clorato) para confirmar a correcta fenótipo. Examine essas colônias para as qualidades desejadas. </li></ol> 4. Os resultados representativos Produzindo peritécios O objectivo é ter um relvado deperitécios sem qualquer micélios ou esporos aparentes. A superfície da cultura aparecerá semelhante a veludo preto (Figura 1). Aos 24 hr após a aplicação de Tween a superfície do ágar deve ter um ligeiro brilho. Se micélios reaparecer, então a placa não pode ter sido raspada suficiente para induzir o desenvolvimento peritécio. Ascósporos de descarga Sempre fornecer na sua ensaio vários blocos de agar a partir da estirpe de tipo selvagem. Se aqueles que não fazer fogo, em seguida, os peritécios são demasiado jovem ou demasiado velho. Se eles são muito antiga, em seguida hifas numerosos irá aparecer sobre a superfície da cultura dando-lhe uma tonalidade esbranquiçada. Se este não for o caso, eles podem ser muito jovens, e no ensaio deve ser tentada de novo em 24 hr. Ocasionalmente, as culturas não cumprir bem, eo experimento terá que ser repetido. Certifique-se de realizar o ensaio nas condições de luz adequadas. Progênie recombinante <p class="jove_content"&gt; Peritécios recombinante deve fazer a maioria de peritécios ao longo da intersecção entre as duas cepas. Nós geralmente processar 10 barbilhoes a partir de uma cruz e, pelo menos, 4-6 daqueles deve ser recombinante. Muito ocasionalmente, uma mutação impede que uma cepa de acasalamento. Se um dos pais tem um fenótipo de crescimento aberrante, em seguida, mais de 2 fenótipos podem estar presentes entre as colónias em MMTS. <img alt="A Figura 1" src="/files/ftp_upload/3895/3895fig1.jpg" /> Figura 1. Estágios de desenvolvimento peritécio em agar de cenoura. H, para a esquerda 72 após a aplicação de Tween. Observação pigmento vermelho e peritécios muito jovem visível como uma grãos negros na superfície. Direito de, a 144 h, peritécios maduros ter se formado. Nota quase completa ausência de micélio superficial em ambas as culturas. <img alt="A Figura 2" src="/files/ftp_upload/3895/3895fig2.jpg" /> Figura ensaio de descarga 2.. A ficha de agar de laranja cenoura é orientada de modo thaesporos t forçosamente descarregadas a partir do peritécios maduro na sua superfície podem acumular-se sobre a superfície da lâmina de vidro. 15 hr tempo de acumulação. <img alt="A Figura 3" src="/files/ftp_upload/3895/3895fig3.jpg" /> Figura 3. Diferenças de crescimento entre Nit + e-NIT colônias em meio MMTS seletivo. As colónias nit-(pontas de seta) são menores. A NIT + de colônias (setas) mostram um crescimento robusto. Fotografia tirada depois de 7 dias.

<ol>
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Não temos nada a divulgar.

F. graminearum está particularmente bem adaptado para o estudo do desenvolvimento corpo de frutificação, devido à disponibilidade de um genoma bem anotado ( <a href="http://mips.helmholtz-muenchen.de/genre/proj/FGDB/">mips.helmholtz-muenchen.de/genre/proj/FGDB /</a> ; 4), e da disponibilidade de uma Affymetrix- baseado microarray 6. Isto facilitou a capacidade de identificar genes importantes para ascósporos descarga 7,11,3. Os métodos apresentados aqui permitirá que o pesquisador se concentrar em um conjunto discreto de estágios de desenvolvimento e funções para a análise genética dos corpos de frutificação de F. graminearum. Os métodos também são facilmente adaptáveis ​​a fungos relacionados que podem ser induzidos a fruta em cultura, e pode ser usado como um padrão para o desenvolvimento de uma variedade de outros tipos de corpo de frutificação fúngicas. A capacidade de avaliar um fenótipo de disparo de esporos podem ser sutis em espécies onde os esporos são pequenos e difíceis de ver, como <em&gt; F. graminearum. A coleção de esporos em uma lâmina limpa facilita a avaliação visual, e avaliação quantitativa, como esporos podem ser lavados fora do slide e quantificada. Algumas espécies de fungos produzir uma mucilagem que rodeia o seu esporo e os esporos não pode ser retirado com água, mas deve ser contados microscopicamente como eles permanecem sobre a lâmina. Nós descobrimos que isso é verdade de Sclerotinia sclerotiorum.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Fusarium graminearum estirpe PH-1 </td><td> Fungal Genetics estoque Center, Kansas </td><td> FGSC 9075 </td></tr><tr><td> Tween 60 </td><td> Sigma-Aldrich </td><td> P1629 </td></tr><tr><td> Agar Czapek Dox do </td><td> Difco, Becton Dickinson </td><td> 233810 </td></tr><tr><td> Cloreto de Potássio </td><td> Sigma-Aldrich </td><td> 255572 </td></tr></tbody></table>

sexual development and ascospore discharge in fusarium graminearum

Fusarium graminearum tornou-se um sistema modelo para estudos em desenvolvimento e patogenicidade de fungos filamentosos. F. graminearum mais facilmente produz corpos de frutificação, chamados peritécios, em ágar cenoura. Peritécios conter inúmeros tipos de tecidos, produzidas em fases específicas do desenvolvimento peritécio. Estes incluem (por ordem de aparecimento) a formação de as iniciais peritécio (que dão origem às hifas ascogenous), a parede exterior, paráfise (micélios estéril que ocupam o centro do peritécio até que o ascos desenvolver), o ascos, e os ascósporos dentro do ascos 14. O desenvolvimento de cada um destes tecidos é separado por aproximadamente 24 horas e tem sido a base em estudos transcriptomic durante o desenvolvimento sexual 12,8. Consulte Hallen et al. (2007) para uma descrição mais completa do desenvolvimento, incluindo fotografias de cada etapa. Aqui, apresentamos os métodos para gerar e colher síncronaly desenvolvimento de gramados de peritécios para estudos temporais de regulação gênica, desenvolvimento e processos fisiológicos. Embora estes métodos são escritos especificamente para ser utilizado com F. graminearum, as técnicas podem ser utilizadas para uma variedade de outros fungos, desde que a frutificação pode ser induzida em cultura e há algum sincronia com o desenvolvimento. Recentemente adaptado este protocolo para estudar o desenvolvimento sexual de F. verticillioides. Embora peritécios indivíduo deve ser colhido manualmente nesta espécie, porque um gramado de desenvolver peritécios não poderia ser induzido, o processo funcionou bem para estudar o desenvolvimento (Sikhakolli e Trail, inédito). A função mais importante de corpos de frutificação de fungos é a dispersão de esporos. Em muitas das espécies de Ascomycota (ascus produzindo fungos), os esporos são disparadas a partir do ascus, devido à geração de pressão de turgescência dentro do ascus, dirigindo ejecção de esporos (e fluido epiplasmic) através doporo na ponta ascus 2,7. Nossos estudos de descarga de ascósporos forçada resultaram no desenvolvimento de um &quot;ensaio de descarga de esporos&quot;, que usamos para triagem de mutações no processo. Apresentamos aqui os detalhes deste ensaio. F. graminearum é homothallic e, portanto, pode formar corpos de frutificação, na ausência de um parceiro compatível. A vantagem de homothallism é que atravessamento não é necessária para gerar descendência homozigótica para um traço particular, uma faceta que facilitou o estudo do desenvolvimento sexual nesta espécie 14,7. No entanto, as estirpes heterotálico ter sido gerados que podem ser utilizados para a passagem 5,9. É também possível para atravessar as estirpes homothallic para se obter mutantes para vários genes na estirpe um 1. Isso é feito por coinoculating uma placa de Petri com 2 estirpes. Ao longo do ponto de encontro, a maioria dos peritécios será recombinante (fornecida uma mutação em uma das estirpes-mãe não inibede cruzamento). Com a idade peritécios, eles exalam ascósporos em massa ao invés de descarregá-los à força. O exsudado de esporos resultante (chamado um cirrhus) senta-se a ponta do peritécio e pode ser facilmente removido para a recuperação de esporos individuais. Aqui nós apresentamos um protocolo para facilitar a identificação de recombinante peritécios ea recuperação da progênie recombinante.

Watch this Scientific Journal Video about Sexual Development and Ascospore Discharge in Fusarium graminearum at JoVE.com

Sexual Development and Ascospore Discharge in Fusarium graminearum

Cruzamentos sexuais e isolamento da progênie recombinante são importantes ferramentas de pesquisa para o fungo filamentoso,<em&gt; Fusarium graminearum</em&gt;, As técnicas necessárias com sucesso realizar estes processos são apresentados.

Desenvolvimento Sexual e descarga de ascósporos em Fusarium graminearum</em

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces ...

sexual-development-and-ascospore-discharge-in-fusarium-graminearum

Research

JoVE Journal

Biology

Sexual Development and Ascospore Discharge in Fusarium graminearum

21.7K visualizações.  Michigan State University. Cruzamentos sexuais e isolamento da progênie recombinante são importantes ferramentas de pesquisa para o fungo filamentoso, Fusarium graminearum, As técnicas necessárias com sucesso realizar estes processos são apresentados.

Vídeo: Sexual Development and Ascospore Discharge in Fusarium graminearum

Gibberella zeae Ascospore Production and Collection for Microarray Experiments.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain quality. F. graminearum also causes stalk and ear rots of maize and is a producer of mycotoxins such as the trichothecenes that contaminate grain and are harmful to humans and livestock (Goswami and Kistler, 2004).

The fungus produces two types of spores. Ascospores, the propagules resulting from sexual reproduction, are the main source of primary infection.  These spores are forcibly discharged from mature perithecia and dispersed by wind (Francl et al 1999). Secondary infections are mainly caused by macroconidia which are produced by asexual means on the plant surface. To study the developmental processes of ascospores in this fungus, a procedure for their collection in large quantity under sterile conditions was required. Our protocol was filmed in order to generate the highest level of information for understanding and reproducibility; crucial aspects when full genome gene expression profiles are generated and interpreted. In particular, the variability of ascospore germination and biological activity are dependent on the prior manipulation of the material. The use of video for documenting every step in ascospore production is proposed in order to increase standardization, complying with the increasingly stringent requirements for microarray analysis. The procedure requires only standard laboratory equipment. Steps are shown to prevent contamination and favor time synchronization of ascospores.

To study the developmental processes of ascospores in Gibberella zeae, a procedure for collection under sterile conditions is filmed in order to generate the highest level of information for protocol description. This should facilitate the reproducibility of the experiment, a crucial aspect when full genome expression profile tests are implemented.

Gibberella zeae produção de ascósporos e Coleta de Experimentos de Microarray.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain ...

Biologia

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo evaluation of putative antitumor compounds.
The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result.
Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures.
Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents.
Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.

In vivo Bioluminescent imagem de tumores mamários Usando Spectrum IVIS

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

Visualizar o coração batendo no Drosophila</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Congelamento e descongelamento, células estaminais embrionárias humanas

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

Indução toxina e Extração de Proteínas de Fusarium Spp. Culturas de Estudos Proteômica

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

Avaliação do desenvolvimento da glândula mamária e Função em modelos de ratos

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

DNA-RNA combinado fluorescente<em&gt; Em situ</em&gt; A hibridização (FISH) para estudar inativação do cromossomo X em Diferenciada Feminino Rato Células-Tronco Embrionárias

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

Microscopia do ciclo de vida sexual por fissão de levedura

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

Gap juncional intercelular comunicação: um biomarcador funcional para avaliar os efeitos adversos de tóxicos e toxinas, e Benefícios para a saúde de produtos naturais

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group developed an intra-molecular FRET sensor to detect the interaction between G&#945; subunits and GPCRs in live cells following agonist stimulation. Here, we detail the protocol for detecting changes in FRET between the &#946;2-adrenergic receptor and the G&#945;s C-terminus peptide upon treatment with 100 &#181;M isoproterenol hydrochloride as previously characterized1. Our FRET sensor is a single polypeptide consisting serially of a full-length GPCR, a FRET acceptor fluorophore (mCitrine), an ER/K SPASM (systematic protein affinity strength modulation) linker, a FRET donor fluorophore (mCerulean), and a G&#945; C-terminal peptide. This protocol will detail cell preparation, transfection conditions, equipment setup, assay execution, and data analysis. This experimental design detects small changes in FRET indicative of protein-protein interactions, and can also be used to compare the strength of interaction across ligands and GPCR-G protein pairings. To enhance the signal-to-noise in our measurements, this protocol requires heightened precision in all steps, and is presented here to enable reproducible execution.

Simple methods to detect the selective activation of G proteins by G protein-coupled receptors remain an outstanding challenge in cell signaling. Here, F&#1255;rster resonance energy transfer (FRET) biosensors have been developed by pairwise tethering a GPCR to G protein peptides to probe conformational changes at controlled concentrations in live cells.

Conformações GPCR G Protein selectivos medida usando FRET Sensores em um Fluorometer Ensaio de células vivas de suspensão

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group ...