Questo lavoro è stato sostenuto da un finanziamento della National Science Foundation (MCB-0.923.794 a FT).

1. Produrre periteci <ol><li> Centro carota agar inoculare 10 in un piatto diam 6,0 centimetri Petri con un ceppo di F. fertile graminearum (Consiglia PH-1). </li><li> Luogo inoculato piatti sotto brillanti luci fluorescenti a temperatura ambiente (18-24 ° C) e lasciar crescere fino micelio ha raggiunto il bordo esterno del piatto Petri (3-5 giorni). Luci fluorescenti per la casa commerciali funzionano bene per stimolare la formazione del corpo fruttifero e di scarico. </li><li> Rimuovere delicatamente il micelio aereo con uno stuzzicadenti sterile. </li><li> Distribuire 1,0 ml 2,5% Tween 60 aq alla superficie con un hockeystick vetro sterile o estremità arrotondata di una bacchetta di vetro sterile. </li><li> Torna piastre alla luce. Non aggiungere Parafilm alle piastre. </li><li> Dopo 24 ore, la superficie delle piastre dovrebbe avere un aspetto brillante. Se miceli riappare, superficie raschiare e riapplicare Tween-60 soluzione. </li><li> Osservare lo sviluppo di periteci la prossima settimana. Le fasi intermedie diperithecium sviluppo può essere raccolto da delicatamente raschiatura della superficie del agar e flash congelati per estrazione di RNA (Figura 1). </li><li> Spore mature si accumulano sul coperchio della piastra di giorno 7. Inizialmente queste saranno visibili sotto un microscopio per dissezione, ma di giorno 9, che si sono accumulati a densità sufficiente in modo da essere visibili a occhio nudo. </li></ol> 2. Ascospore scarico Assay <ol><li> Il giorno 6 dopo l&#39;applicazione della soluzione di Tween, tagliare un cerchio di diametro 1 cm dal agar con un piralide legno. In alternativa, un bisturi può essere usato per rimuovere un segmento di dimensioni analoghe. Il cerchio può essere tagliati a metà e ciascuna metà posta su un vetrino per microscopio. </li><li> Orientare i pezzi così la superficie contenente i corpi fruttiferi è perpendicolare alla superficie della slitta, e le spore vengono sparati lungo la lunghezza della slitta. </li><li> Mettere il vetrino in una camera umida per tutta la notte sotto le luci. Le spore si accumulano sullala slitta ed essere visibile ad occhio nudo la mattina seguente (figura 2). </li><li> Spore accumulate possono essere lavati dal vetrino con acqua e quantificati se lo si desidera. </li><li> Potenziali inibitori scarico ascospora può essere saggiata aggiungendoli alla parte posteriore del blocco agar durante l&#39;assemblaggio del saggio. Alcuni inibitori del canale ionico che riducono scarico sono stati precedentemente esaminati con questa tecnica 15. </li></ol> 3. Generazione di Progeny ricombinante <ol><li> Avviare attraversare inoculando agar di carote con due ceppi di F. graminearum (uno Nit + e uno nit-), ponendo i punti di inoculazione su lati opposti della piastra di Petri. </li><li> Dopo aver riempito la ife superficie, raschiare porzione aerea e applicare Tween-60 soluzione come descritto sopra. Utilizzare un marcatore notare sul lato della piastra Petri dove ife incontrano. </li><li> Torna culture alla luce e incubare fino periteci hanno sviluppato, e hanno cirrhi esserepistola a formare dal periteci lungo l&#39;intersezione tra le due culture. </li><li> Sotto il microscopio da dissezione, mettono in visualizzare il periteci lungo l&#39;intersezione delle due culture. L&#39;utilizzo di un pin insetto, sterilizzato e immerso in acqua sterile, toccare un cirrhus lungo questo confine con la punta del perno. L&#39;umidità del perno dovrebbe consentire alle cirrhus ad attaccarsi al perno. </li><li> Lavare la punta del perno con i cirrhus su di esso in 200 microlitri di acqua sterile in un tubo Eppendorf a lavare le spore. </li><li> Flame il perno, si inumidire di nuovo e scegliere un altro cirrhus. Molto 10-15 cirrhi, e posizionare ciascuno in un tubo separato di acqua. </li><li> Brevemente i tubi vortex in sospensione cirrhi. </li><li> Rimuovere 60 pl di sospensione di spore con una pipetta e posto sul terreno MMTS 1 in una capsula Petri cm 9. Stendere con una mazza da hockey sterile. La sospensione di spore rimanente può essere conservato a 4 ° C per una settimana e ripiastrate se necessario. </li><li> IncubaTE a temperatura ambiente (luce non necessaria) per 3-5 giorni fino colonie molto piccole (Figura 3). Ci saranno due tipi di fenotipi tra le colonie. I nit-colonie avrà ife rada di colonie + il NIT. Cirrhi da ricombinante periteci mostrerà entrambi i fenotipi di colonie in proporzione approssimativamente uguale. Gettare le piastre con colonie di solo un fenotipo singolo su di loro. Si noti che a volte più di due fenotipi risultare a causa della segregazione di altri fattori. </li><li> Spostare colonie V8 o agar carota per lo stoccaggio. Colonie di test su Agar Dox Czapek (le nit di ceppi non cresce su Dox Czapek) e su PDA con clorato (0,5 M clorato di potassio, i ceppi + Nit non crescono su PDA con Clorato) per confermare il fenotipo corretta. Esaminare queste colonie per le qualità desiderate. </li></ol> 4. Risultati rappresentativi Produrre periteci L&#39;obiettivo è quello di avere un prato diperiteci senza miceli o spore apparenti. La superficie della coltura sarà simile a velluto nero (Figura 1). A 24 ore dopo l&#39;applicazione di Tween superficie del agar dovrebbe avere una leggera lucentezza. Se miceli riapparire, poi la targa non può essere stato raschiato a sufficienza per indurre lo sviluppo perithecium. Ascospore di scarico Fornire sempre nel vostro test diversi blocchi di agar dal ceppo di tipo selvatico. Se questi non vengono attivati, quindi sia i periteci sono troppo giovani o troppo vecchi. Se sono troppo vecchio, quindi ife numerosi apparirà sulla superficie della cultura dando una tonalità biancastra. Se questo non è il caso, essi possono essere troppo giovane, e il saggio deve essere nuovamente processato in 24 ore. Di tanto in tanto, le culture non si scaricano bene, e l&#39;esperimento dovrà essere ripetuto. Assicurati di eseguire il test in base alle condizioni di luce adeguate. Progenie ricombinante <p class="jove_content"Periteci&gt; ricombinante dovrebbero costituire la maggior parte dei periteci lungo l&#39;intersezione tra i due ceppi. Noi di solito processo 10 cirrhi da una croce e almeno 4-6 di questi dovrebbe essere ricombinante. Molto raramente, una mutazione impedisce che un ceppo di accoppiamento. Se uno dei genitori ha un fenotipo aberrante crescita, quindi più di 2 fenotipi possono essere presenti tra le colonie MMTS. <img alt="Figura 1" src="/files/ftp_upload/3895/3895fig1.jpg" /> Figura 1. Fasi di sviluppo perithecium su agar carota. Sinistra, 72 h dopo l&#39;applicazione di Tween. Nota pigmento rosso e periteci giovanissimo visibile come grani nere sulla superficie. A destra, a 144 h, periteci maturi si sono formate. Nota quasi completa assenza di miceli superficiale in entrambe le culture. <img alt="Figura 2" src="/files/ftp_upload/3895/3895fig2.jpg" /> Figura 2. Scarico saggio. L&#39;arancio spina agar carota è orientata in modo thaspore t forzatamente scaricate dal periteci maturo sulla sua superficie può accumularsi sulla superficie del vetrino. 15 ore tempo di accumulo. <img alt="Figura 3" src="/files/ftp_upload/3895/3895fig3.jpg" /> Figura 3. Le differenze di crescita tra + e Nit nit-colonie sul terreno selettivo MMTS. I nit-colonie (punte di freccia) sono più piccoli. Le colonie + NIT (frecce) mostrano una crescita robusta. Fotografia scattata dopo 7 giorni.

<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. -. H., Guldener, U., 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=Gene+expression+shifts+during+perithecium+development+in+Gibberella+zeae+(anamorph+Fusarium+graminearum),+with+particular+emphasis+on+ion+transport+proteins.">Gene expression shifts during perithecium development in Gibberella zeae (anamorph Fusarium graminearum), with particular emphasis on ion transport proteins.</a> Fungal Genetics and Biology. 44, 1146-1156 (2007).</li><li>Lee, J., Lee, T., Lee, Y. -. W., Yun, S. -. H., 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=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. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+gene,+ROA,+is+required+for+proper+morphogenesis+and+discharge+of+ascospores+in+Gibberella+zeae.">A novel gene, ROA, is required for proper morphogenesis and discharge of ascospores in Gibberella zeae.</a> Eukaryotic Cell. 9, 1495-1503 (2010).</li><li>Qi, W., Kwon, C., 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=Microarray+analysis+of+transcript+accumulation+during+perithecium+development+in+Gibberella+zeae+(anamorph+Fusarium+graminearum).">Microarray analysis of transcript accumulation during perithecium development in Gibberella zeae (anamorph Fusarium graminearum).</a> Molecular Genetics and Genomics. , 276-287 (2006).</li><li>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=Fungal+cannons:+explosive+spore+discharge+in+the+Ascomycota.">Fungal cannons: explosive spore discharge in the Ascomycota.</a> FEMS Microbiol. 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>

Non abbiamo nulla da rivelare.

F. graminearum è particolarmente adatto per lo studio di sviluppo fruttificazione corpo grazie alla disponibilità di una ben annotato genoma ( <a href="http://mips.helmholtz-muenchen.de/genre/proj/FGDB/">mips.helmholtz-muenchen.de/genre/proj/FGDB /</a> , 4), e la disponibilità di un Affymetrix, basato microarray 6. Questo ha facilitato la capacità di identificare i geni importanti per lo scarico ascospore 7,11,3. I metodi qui presentati permetteranno al ricercatore di concentrarsi su un insieme discreto di stadi di sviluppo e le funzioni per l&#39;analisi genetica dei corpi fruttiferi di F. graminearum. I metodi sono inoltre facilmente adattabili a funghi correlati che possono essere indotte a frutta in coltura, e può essere utilizzato come standard per lo sviluppo in una varietà di tipi fruttiferi altri fungine corpo. La capacità di valutare un fenotipo spore di cottura può essere sottile nelle specie in cui spore sono piccole e difficili da vedere, come <em&gt; F. graminearum. La raccolta di spore su un vetrino pulito facilita sia la valutazione visiva, e la valutazione quantitativa, in quanto le spore possono essere lavati dal vetrino e quantificato. Alcune specie fungine producono una mucillagine che circonda la loro spore e le spore non possono essere risciacquata, ma devono essere contati al microscopio che restano sulla slitta. Abbiamo trovato che questo è vero di Sclerotinia sclerotiorum.

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Fusarium graminearum ceppo PH-1 </td><td> Fungina Genetica archivi Center, Kansas </td><td> FGSC 9075 </td></tr><tr><td> Tween 60 </td><td> Sigma-Aldrich </td><td> P1629 </td></tr><tr><td> Dox Agar Czapek di </td><td> Difco, Becton Dickinson </td><td> 233810 </td></tr><tr><td> Clorato di potassio </td><td> Sigma-Aldrich </td><td> 255572 </td></tr></tbody></table>

sexual development and ascospore discharge in fusarium graminearum

Fusarium graminearum è diventato un sistema modello per gli studi nello sviluppo e la patogenicità dei funghi filamentosi. F. graminearum più facilmente produce corpi fruttiferi, detti periteci, su agar carota. Periteci contengono numerosi tipi di tessuti, prodotti in determinate fasi dello sviluppo perithecium. Questi includono (in ordine di apparizione) formazione delle iniziali perithecium (che dà luogo a ife ascogenous), il muro esterno, paraphyses (miceli sterili che occupano il centro della perithecium fino al ASCI sviluppo), l&#39;ASCI, e le ascospore all&#39;interno aschi 14. Lo sviluppo di ciascuno di questi tessuti è separato da circa 24 ore ed è stata la base di studi trascrittomica durante lo sviluppo sessuale 12,8. Fare riferimento alla Hallen et al. (2007) per una descrizione più approfondita di sviluppo, comprese le fotografie di ogni tappa. Qui vi presentiamo i metodi per la generazione e la raccolta sincronamente lo sviluppo di prati di periteci per gli studi temporali di regolazione genica, sviluppo e processi fisiologici. Sebbene questi metodi sono scritti specificamente per essere utilizzato con F. graminearum, le tecniche possono essere utilizzate per una varietà di altri funghi, purché fruttificazione può essere indotta in coltura e vi è una certa sincronia di sviluppo. Di recente abbiamo adattato questo protocollo per studiare lo sviluppo sessuale di F. verticillioides. Anche se periteci individuo deve essere raccolte a mano in questa specie, a causa di un prato in via di sviluppo periteci non poteva essere indotta, il processo ha funzionato bene per lo studio dello sviluppo (Sikhakolli e Trail, inedito). La funzione più importante di corpi fruttiferi fungini è la dispersione delle spore. In molte delle specie di Ascomycota (asco produrre funghi), spore vengono sparati da asco, a causa della generazione di pressione all&#39;interno del turgore asco, guida espulsione di spore (e fluido epiplasmic) attraverso l&#39;pori nella punta asco 2,7. I nostri studi di scarico forzato ascospore hanno portato allo sviluppo di un &quot;test di scarico spore&quot;, che usiamo per lo screening di mutazioni nel processo. Qui vi presentiamo i dettagli di questo test. F. graminearum è homothallic, e quindi possono formare corpi fruttiferi in assenza di un partner compatibile. Il vantaggio di homothallism è che la traversata non è necessario generare prole omozigote per un carattere particolare, un aspetto che ha facilitato lo studio dello sviluppo sessuale in questa specie 14,7. Tuttavia, heterothallic ceppi sono stati generati che possono essere utilizzati per l&#39;attraversamento 5,9. È anche possibile attraversare ceppi homothallic ottenere mutanti dei geni diversi in un unico ceppo 1. Questo viene fatto coinoculating un piatto Petri con 2 ceppi. Lungo il punto di incontro, la maggioranza dei periteci sarà ricombinante (fornita una mutazione in uno dei ceppi madri non inibisceincroci). Con l&#39;età periteci, trasudano ascospore in massa, invece di forza li scarica. L&#39;essudato spore risultante (chiamato cirrhus) si trova sulla punta del perithecium e può essere facilmente rimossa per il recupero di spore individuali. Qui vi presentiamo un protocollo per facilitare l&#39;identificazione dei periteci ricombinante e il recupero della progenie ricombinante.

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

Croci sessuali e isolamento della progenie ricombinante sono strumenti di ricerca importanti per il fungo filamentoso,<em&gt; Fusarium graminearum</em&gt;, Le tecniche necessarie successo svolgere questi processi sono presentati.

Sviluppo sessuale e scarico in ascospore 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 Views.  Michigan State University. Croci sessuali e isolamento della progenie ricombinante sono strumenti di ricerca importanti per il fungo filamentoso, Fusarium graminearum, Le tecniche necessarie successo svolgere questi processi sono presentati.

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 ascospore di produzione e di raccolta di esperimenti di microarray.

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

Ricerca

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 Imaging dei tumori mammari che usano lo spettro 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.

Visualizzare il cuore pulsante di 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 scongelamento cellule staminali embrionali umane

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.

Tossina induzione ed estrazione di proteine ​​da Fusarium Spp. Culture per studi di proteomica

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.

Valutazione dello Sviluppo ghiandola mammaria e funzione in modelli murini

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 combinato fluorescente<em&gt; In situ</em&gt; Ibridazione (FISH) per studiare inattivazione del cromosoma X nelle cellule staminali embrionali di topo differenziata Femminile

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 di lievito di fissione del ciclo di vita sessuale

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.

Spazi di giunzione intercellulare di comunicazione: un biomarker funzionale per valutare gli effetti avversi dei tossici e tossine, e benefici per la salute di prodotti naturali

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.

Conformazioni GPCR g di proteine-selettivi misurata utilizzando FRET sensori in un Fluorometer Assay Live Cell Suspension

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