Este trabajo fue apoyado por una beca de la National Science Foundation (MCB-0923794 a FT).

1. La producción de peritecios <ol><li> Inocular el Centro de zanahoria agar 10 en un 6,0 diámetro cm plato de Petri con una cepa fértil de F. graminearum (se recomienda PH-1). </li><li> Colocar inocularon platos bajo luces fluorescentes brillantes a temperatura ambiente (18-24 ° C) y permitir a crecer hasta micelio ha alcanzado el borde exterior de la placa de Petri (3-5 días). Las luces fluorescentes comerciales domésticos funcionar bien para estimular la formación de cuerpo fructífero y descarga. </li><li> Suavemente eliminar los micelios aéreos con un palillo estéril. </li><li> Distribuir 1,0 ml de 2,5% de Tween 60 acuoso a la superficie con un palo de hockey de vidrio estéril o extremo redondeado de una varilla de vidrio estéril. </li><li> Volver placas a la luz. No agregue Parafilm a las placas. </li><li> Después de 24 horas, la superficie de las placas deben tener un aspecto brillante. Si vuelve a aparecer el micelio, la superficie de raspado y volver a aplicar la solución de Tween-60. </li><li> Observar el desarrollo de peritecios durante la próxima semana. Las etapas intermedias dedesarrollo peritecio pueden ser cosechadas raspando suavemente la superficie del agar y flash congelado para extracción de RNA (Figura 1). </li><li> Esporas maduras se acumulan en la tapa de la placa por día 7. Inicialmente éstos sólo serán visibles bajo un microscopio de disección, pero por día 9, se han acumulado a la densidad suficiente como para ser visible a simple vista. </li></ol> 2. Ascosporas ensayo de descarga <ol><li> El día 6 después de la aplicación de la solución de Tween, cortar un círculo de 1 cm de diámetro del agar con un perforador de madera. Alternativamente, un bisturí puede ser utilizado para cortar un segmento de tamaño similar. El círculo puede ser cortado por la mitad y cada mitad coloca en un portaobjetos de vidrio. </li><li> Orient las piezas manera que la superficie que contiene los cuerpos fructíferos es perpendicular a la superficie de la corredera, y las esporas se disparan a lo largo de la corredera. </li><li> Colocar el portaobjetos en una cámara de humedad durante la noche bajo las luces. Las esporas se acumulará sobrela corredera y ser visibles para el ojo desnudo a la mañana siguiente (Figura 2). </li><li> Esporas acumuladas se pueden lavar fuera de la diapositiva con agua y se cuantificó si se desea. </li><li> Potenciales inhibidores de descarga de ascosporas se puede ensayar por adición a la parte trasera del bloque de agar durante el montaje del ensayo. Algunos inhibidores de los canales iónicos que reducen la descarga han sido previamente ensayados con esta técnica 15. </li></ol> 3. Generación de progenie recombinante <ol><li> Inicia cruzada mediante la inoculación de agar zanahoria con dos cepas de F. graminearum (una Nit + y una nits), colocando los puntos de inoculación en los lados opuestos de la placa de Petri. </li><li> Una vez que las hifas han llenado la superficie, raspar parte aérea y aplicar de Tween-60 solución como se describe anteriormente. Use un marcador para observar en el lado de la placa de Petri donde las hifas se encuentran. </li><li> Volver culturas a la luz y se incuba hasta peritecios se han desarrollado, y que han cirrhiarma para formar a los peritecios lo largo de la intersección entre las dos culturas. </li><li> Bajo el microscopio de disección, traer a la vista los peritecios lo largo de la intersección de las dos culturas. Utilizando un pasador de insectos, se esteriliza y se sumerge en agua estéril, toque una cirrhus a lo largo de este borde ligeramente con la punta de la clavija. La humedad en el pasador debe permitir que las cirrhus a pegarse a la clavija. </li><li> Enjuague la punta de la espiga con los cirrhus en que de cada 200 l de agua estéril en un tubo Eppendorf de separar las esporas. </li><li> Llama la clavija, se humedece de nuevo y elegir otro cirrhus. Elige 10 a 15 cirrhi, y colocar cada uno en un tubo separado de agua. </li><li> Tubos, agitar brevemente que contiene suspendidas cirrhi. </li><li> Retirar 60 l de la suspensión de esporas con una pipeta y el lugar en un medio MMTS 1 en un 9 cm de placa de Petri. Extender con un palo de hockey estéril. La suspensión de esporas restante puede ser almacenado a 4 ° C durante hasta una semana y replated si es necesario. </li><li> IncubaTE a temperatura ambiente (la luz no es necesario) durante 3-5 días hasta que las colonias muy pequeñas aparecen (Figura 3). Habrá dos tipos de fenotipos entre las colonias. Las colonias de nit tendrá hifas más escasas que las colonias + Nit. Cirrhi recombinante de peritecios se muestran ambos fenotipos de colonias en proporción aproximadamente igual. Desechar las placas con colonias de sólo un fenotipo en ellos. Tenga en cuenta que a veces, más de dos fenotipos resultar debido a la segregación de otros factores. </li><li> Mueva las colonias de agar V8 o de zanahoria para el almacenamiento. Colonias de prueba en agar Czapek Dox (las cepas de nit-no crece en Czapek Dox) y el PDA con el clorato (0,5 clorato de potasio M; las cepas Nit + no va a crecer en la PDA con clorato) para confirmar el fenotipo correcta. Examine estas colonias de las cualidades deseadas. </li></ol> 4. Los resultados representativos La producción de peritecios El objetivo es tener un césped deperitecios sin micelios o esporas aparentes. La superficie de la cultura tendrá un aspecto similar al terciopelo negro (Figura 1). A las 24 h después de la aplicación de Tween la superficie del agar debe tener un ligero brillo. Si vuelve a aparecer el micelio, a continuación, la placa no puede haber sido raspada suficiente para inducir el desarrollo peritecios. Ascosporas de descarga Siempre dio en su ensayo de varios bloques de agar de la cepa de tipo salvaje. Si no lo hacen los bomberos, entonces o bien los peritecios son demasiado jóvenes o demasiado viejos. Si son demasiado viejos, y luego las hifas numerosos aparecerá sobre la superficie de la cultura le da un tono blanquecino. Si este no es el caso, pueden ser demasiado joven, y el ensayo debe ser juzgado de nuevo en 24 horas. Ocasionalmente, los cultivos no se descargan bien, y el experimento tendrá que ser repetido. Asegúrese de realizar el ensayo en las condiciones de luz adecuadas. Progenie recombinante <p class="jove_content"Peritecios&gt; recombinante deben constituir la mayoría de los peritecios lo largo de la intersección entre las dos cepas. Por lo general, procesar 10 cirrhi de una cruz y por lo menos 4.6 de los que debe ser recombinante. Muy de vez en cuando, una mutación impide que una cepa de apareamiento. Si uno de los padres tiene un fenotipo de crecimiento aberrante, entonces, más de 2 fenotipos pueden estar presentes entre las colonias en MMTS. <img alt="Figura 1" src="/files/ftp_upload/3895/3895fig1.jpg" /> Figura 1. Etapas del desarrollo de peritecios en agar zanahoria. H Izquierda, de 72 años después de la aplicación de Tween. Nota pigmento rojo y peritecios muy jóvenes visible como algunos granos negros en la superficie. Derecho, a 144 h, peritecios maduros se han formado. Tenga en cuenta casi completa ausencia de micelio superficial en ambas culturas. <img alt="Figura 2" src="/files/ftp_upload/3895/3895fig2.jpg" /> Figura 2. Descarga ensayo. El tapón de color naranja zanahoria agar está orientado de manera Thaesporas t fuerza descargados del peritecios maduro en su superficie se puede acumular en la superficie de la lámina de vidrio. 15 horas tiempo de acumulación. <img alt="Figura 3" src="/files/ftp_upload/3895/3895fig3.jpg" /> Figura 3. Las diferencias de crecimiento entre Nit + y nit de colonias en medio MMTS selectiva. Las colonias de nit (puntas de flecha) son más pequeños. Las colonias Nit + (flechas) muestran un crecimiento robusto. Fotografía tomada después de 7 días.

<ol>
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H., Proctor, R. H., Lee, T., Plattner, R. D., Lu, S. -. W., Turgeon, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+and+complementation+of+the+mating+type+(MAT)+locus+of+the+wheat+head+blight+pathogen+Gibberella+zeae.">Deletion and complementation of the mating type (MAT) locus of the wheat head blight pathogen Gibberella zeae.</a> Applied and Environmental Microbiology. 70, 2437-2444 (2004).</li><li>Gueldener, U., Seong, K. -. Y., Boddu, J., Cho, S., Trail, F., Xu, J. -. R., Adam, G., Mewes, H. -. W., Muehlbauer, G. J., Kistler, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Fusarium+graminearum+Affymetrix+GeneChip+For+profiling+fungal+gene+expression+in+vitro+and+in+planta.">Development of a Fusarium graminearum Affymetrix GeneChip For profiling fungal gene expression in vitro and in planta.</a> Fungal Genetics and Biology. 43, 316-325 (2006).</li><li>Hallen, H., Trail, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+L-Type+calcium+ion+channel+Cch1+affects+ascospore+discharge+and+mycelial+growth+in+the+filamentous+fungus+Gibberella+zeae+(anamorph+Fusarium+graminearum).">The L-Type calcium ion channel Cch1 affects ascospore discharge and mycelial growth in the filamentous fungus Gibberella zeae (anamorph Fusarium graminearum).</a> Eukaryotic Cell. 7, 415-424 (2008).</li><li>Hallen, H., Huebner, M., Shiu, S. -. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shifting+fungal+reproductive+mode+by+manipulation+of+mating+type+genes:+obligatory+heterothallism+of+Gibberella+zeae.">Shifting fungal reproductive mode by manipulation of mating type genes: obligatory heterothallism of Gibberella zeae.</a> Molecular Microbiology. 50, 145-152 (2003).</li><li>Klittich, C. J. R., Leslie, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrate+reduction+mutants+of+Fusarium+moniliforme+(Gibberella+fujikuoi.">Nitrate reduction mutants of Fusarium moniliforme (Gibberella fujikuoi.</a> Genetics. 118, 417-423 (1988).</li><li>Min, K., Lee, J., Kim, J. -. C., Kim, S. G., Kim, Y. H., Vogel, S., Trail, F., Lee, Y. -. 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Lett. 276, 12-18 (2007).</li><li>Trail, F., Common, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perithecial+development+by+Gibberella+zeae:+a+light+microscopy+study.">Perithecial development by Gibberella zeae: a light microscopy study.</a> Mycologia. 92, 130-138 (2000).</li><li>Trail, F., Xu, H., Loranger, R., Gadoury, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+and+environmental+aspects+of+ascospore+discharge+in+Gibberella+zeae+(anamorph+Fusarium+graminearum.">Physiological and environmental aspects of ascospore discharge in Gibberella zeae (anamorph Fusarium graminearum.</a> Mycologica. 94, 181-189 (2002).</li></ol>

No tenemos nada que revelar.

F. graminearum está particularmente bien adaptado para el estudio del desarrollo de la fructificación cuerpo debido a la disponibilidad de un genoma bien anotado ( <a href="http://mips.helmholtz-muenchen.de/genre/proj/FGDB/">mips.helmholtz-muenchen.de/genre/proj/FGDB /</a> ; 4), y la disponibilidad de un Affymetrix- base de microarrays 6. Esto ha facilitado la capacidad de identificar genes importantes para la descarga de ascosporas 7,11,3. Los métodos que aquí se presentan permitirá a los investigadores a centrarse en un conjunto discreto de las etapas de desarrollo y las funciones para el análisis genético de los cuerpos fructíferos de F. graminearum. Los métodos también son fácilmente adaptables a los hongos relacionados que pueden ser inducidos a las frutas en cultivo, y puede ser utilizado como un estándar para el desarrollo en una variedad de otros tipos de hongos fructificación cuerpo. La capacidad de evaluar un fenotipo de cocción de esporas pueden ser sutiles en las especies donde las esporas son pequeños y difíciles de ver, tales como <em&gt; F. graminearum. La colección de esporas en un portaobjetos limpio facilita tanto la evaluación visual, y la evaluación cuantitativa, ya que las esporas pueden ser lavados fuera de la diapositiva y su cuantificación. Algunas especies de hongos producen un mucílago que rodea a la espora y las esporas no se pueden enjuagar, pero deben ser contados al microscopio y cuando se mantengan en la diapositiva. Hemos encontrado que esto es cierto de Sclerotinia sclerotiorum.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Fusarium graminearum cepa PH-1 </td><td> Hongos Genética Stock 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 del </td><td> Difco, Becton Dickinson </td><td> 233810 </td></tr><tr><td> Clorato de potasio </td><td> Sigma-Aldrich </td><td> 255572 </td></tr></tbody></table>

sexual development and ascospore discharge in fusarium graminearum

Fusarium graminearum se ha convertido en un sistema modelo para estudios en el desarrollo y la patogenicidad de los hongos filamentosos. F. graminearum más fácilmente produce cuerpos fructíferos, llamados peritecios, en agar zanahoria. Peritecios contienen numerosos tipos de tejidos, producidos en etapas específicas de desarrollo peritecios. Estos incluyen (en orden de aparición) la formación de las iniciales peritecio (que dan lugar a las hifas ascogenous), la pared exterior, Parafisos (micelio estéril que ocupan el centro de la peritecio hasta el ascos desarrollo), de los ascos y ascosporas dentro de los ascos 14. El desarrollo de cada uno de estos tejidos está separado por aproximadamente 24 horas y ha sido la base de los estudios transcriptomic durante el desarrollo sexual de 12,8. Consulte Hallen et al. (2007) para una descripción más detallada del desarrollo, incluidas las fotografías de cada etapa. Aquí se presentan los métodos para la generación y aprovechamiento síncronomente el desarrollo de jardines de peritecios para estudios temporales de regulación de los genes, el desarrollo y los procesos fisiológicos. Aunque estos métodos están escritos específicamente para ser utilizado con F. graminearum, las técnicas se pueden utilizar para una variedad de otros hongos, siempre que la fructificación se puede inducir en el cultivo y hay una cierta sincronía con el desarrollo. Recientemente hemos adaptado este protocolo para estudiar el desarrollo sexual de F. verticillioides. A pesar de peritecios individuo debe ser recogidas a mano en esta especie, ya que un césped de desarrollar peritecios no podía ser inducido, el proceso funcionó bien para estudiar el desarrollo (Sikhakolli y Trail, sin publicar). La función más importante de los cuerpos fructíferos de hongos es la dispersión de las esporas. En muchas de las especies de Ascomycota (asca hongos productores), las esporas se tiran desde el asca, debido a la generación de presión de turgencia dentro del asca, la conducción de eyección de esporas (y fluido epiplasmic) a través delporo en la punta asca 2,7. Nuestros estudios de la descarga de ascosporas por la fuerza han dado como resultado el desarrollo de un &quot;ensayo de descarga de esporas&quot;, que utilizamos para la detección de mutaciones en el proceso. Aquí se presentan los detalles de este ensayo. F. graminearum es homotálica, y por lo tanto pueden formar cuerpos fructíferos en ausencia de una pareja compatible. La ventaja de homothallism es que el cruce no es necesario para generar descendencia homocigota para un rasgo particular, una faceta que ha facilitado el estudio del desarrollo sexual en esta especie 14,7. Sin embargo, las cepas heterotálica se han generado que se puede utilizar para el cruce de 5,9. También es posible cruzar cepas homotálicos para obtener mutantes de varios genes en una cepa 1. Esto se hace por coinoculating una placa de Petri con 2 cepas. A lo largo del punto de encuentro, la mayoría de peritecios será recombinante (siempre una mutación en una de las cepas progenitoras no inhibepolinización cruzada). A medida que los peritecios, que exudan las ascosporas en masa en lugar de forzar a la descarga. El exudado de esporas resultante (denominado un cirrhus) se sienta en la punta de la peritecio y se puede quitar fácilmente para la recuperación de esporas individuales. Aquí se presenta un protocolo para facilitar la identificación de los peritecios recombinante y la recuperación de la progenie 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

Cruces sexuales y el aislamiento de la progenie recombinante son importantes herramientas de investigación para el hongo filamentoso,<em&gt; Fusarium graminearum</em&gt; Las técnicas necesarias realizar con éxito estos procesos se presentan.

Desarrollo Sexual y descarga en las ascosporas 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

21.8K vistas.  Michigan State University. Cruces sexuales y el aislamiento de la progenie recombinante son importantes herramientas de investigación para el hongo filamentoso, Fusarium graminearum Las técnicas necesarias realizar con éxito estos procesos se presentan.

Video: 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 ascósporas de producción y de recolección de experimentos de microarrays.

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

Investigación

Biología

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.

En vivo bioluminiscente de imágenes de tumores de mama mediante el espectro de 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 el corazón latiendo en Drosophila</em

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

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

La cultura y el mantenimiento de células estaminales embrionarias humanas

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

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.

Congelación y descongelación células estaminales embrionarias 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.

La toxina de inducción y extracción de proteínas a partir de Fusarium Spp. Los cultivos para los estudios proteómicos

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.

Evaluación de la glándula mamaria de desarrollo y función en modelos de ratón

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.

ADN-ARN combinado fluorescente<em&gt; In situ</em&gt; La hibridación (FISH) para el Estudio de inactivación del cromosoma X en las células madre embrionarias de ratón Diferenciada Mujer

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.

Microscopía de la levadura de fisión del ciclo de vida sexual

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.

Brecha ocasiones intercelular de comunicación: un biomarcador funcional para evaluar los efectos adversos de las sustancias tóxicas y toxinas, y de Salud Beneficios de Productos Naturales

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