Questo protocollo è stato sviluppato nei laboratori di Jane Dodd, Thomas Jessell e Andrew Lumsden. Molte persone, tra cui Konrad Basler, Ann Calof, Thomas Edlund, Phil Hamilton, Domna Karagogeos, Ariel Ruiz i Altaba e Toshiya Yamada determinato come espianti cultura del midollo spinale nel collagene. Marysia Placzek e Marc Tessier-Lavigne sperimentato la tecnica di usare la crescita degli assoni da espianti in vitro come mezzo per identificare molecole di guida degli assoni. Lavoro in laboratorio Butler è sostenuto da finanziamenti della March of Dimes e R01 NS063999 dal NIH / NINDS.

Parte 1: Preparazione di aggregati di cellule COS transfettati con gocce <ol><li> COS-7 di semi (COS), le cellule in capsule di Petri da 35 mm. Quando raggiungono l&#39;80% confluenza, 1μg trasfezione del plasmide di espressione nelle cellule utilizzando Lipofectamine2000 secondo il protocollo del produttore s. </li><li> Per preparare appeso gocce, aspirare il terreno di trasfezione e risciacquare le cellule COS transfettate con 1 ml di soluzione tamponata 1x fosfato (PBS). Il trattamento di cellule con 0,5 ml di medio enzima cellulare senza dissociazione per 15 minuti. Aggiungere 1 ml di una soluzione di Opti-MEM + 1x Penicillina / Streptomicina / Glutammina (P / S / G) + 10% siero fetale bovino (FBS) per arrestare la reazione. </li><li> Triturare le cellule per rimuoverli dalla superficie del piatto cultura e trasferire in una provetta da 15 ml conica. Spin per 2 minuti a 2000K per agglomerare le cellule. Rimuovere il surnatante e risospendere pellet in 100μl di Opti-MEM + 1x P / S / G + 10% FBS. </li><li> Spot più gocce 20μl all&#39;interno di un coperchio piatto 35 millimetri cultura, capovolgere il coperchio e posto in cima al fondo del piatto e lasciare in un incubatore a 37 ° C per diverse ore fino a quando le cellule aggregato. </li></ol> Parte 2: Preparazione del midollo spinale dorsale Espianti <ol><li> Sezionare embrioni di ratto E11 dall&#39;utero della madre e tenere sul ghiaccio in L15 medio fino a quando necessario. </li><li> Con un ago di tungsteno affilato, rimuovere una sezione 4-6 segmento dal tronco di ogni embrione, dalla regione immediatamente al di sotto degli arti anteriori gemma. Utilizzando una pipetta di plastica, raccogliere i pezzi di tessuto in un pozzetto di un 4-Nunc ben piatto e tenere in ghiaccio. </li><li> Quando tutti i segmenti embrione sono stati sezionati, utilizzare la pipetta di plastica per trasferire i pezzi in una soluzione di 1ml L15 + 1mg dispasi in un secondo pozzo del piatto Nunc. Incubare a temperatura ambiente per non più di 5 minuti. Non più di incubare i pezzi di tessuto con dispasi. </li><li> Durante l&#39;incubazione, aggiungere 0,5 ml di siero inattivato con il calore di capra normale (HIGS) a circa 10 ml di L15 in una piastra di Petri e mescolare. Trasferire 1 ml di questa soluzione in un terzo pozzo del piatto Nunc e trasferire i pezzi di tessuto in questa soluzione quando l&#39;incubazione è finita. Tenere su ghiaccio. Nota: la dissezione successiva sarà più facile se i pezzi di tessuto sono autorizzati a &quot;riposo&quot; in ghiaccio per un&#39;ora. </li></ol> Parte 3: Collagene Priming <ol><li> Aggiungere 40μl 10x terreno minimo essenziale al collagene 360μl, mescolare rapidamente e accuratamente sfogliando o brevemente il tubo vortex. Spin down rapidamente in un picofuge. La soluzione sarà giallo. Tenere sul ghiaccio il più possibile. </li><li> Aggiungere abbastanza bicarbonato di sodio 0,8 M (NaHCO 3) per trasformare la soluzione collagene leggermente arancione (vedi nota sotto). Ancora una volta, mescolare rapidamente e accuratamente sfogliando, e spin down brevemente in una picofuge. Se la soluzione rimane rosa dopo la miscelazione, aver aggiunto troppo NaHCO 3 e sarà necessario ricominciare da capo con un tubo fresco di collagene. </li><li> A questo punto la soluzione di collagene è innescato. Rimarrà liquido sul ghiaccio, ma si solidificano in circa 5 minuti (girando rosa) quando portati a temperatura ambiente. </li><li> 20μl posto del collagene in ciascun pozzetto di una 4-ben piatto Nunc. Utilizzando la punta della pipetta, si sviluppa il collagene per formare una piccola &quot;pad&quot;. Lasciate che il collagene fissato a temperatura ambiente. </li></ol> Nota: la quantità esatta di NaHCO 3 dovranno essere titolati per ogni lotto di collagene. Inizio bassa (11μl) e quindi aggiungere 0,5-1ml gradualmente fino a quando la soluzione è una tonalità leggermente di arancio. La quantità &quot;giusta&quot; è di solito 1UL inferiore alla più piccola quantità di NaHCO 3, che farebbe diventare la rosa collagene. La quantità di NaHCO 3 non scala linearmente su o giù. Parte 4: Dissezione di dorsali Espianti midollo spinale e posizionamento di loro con aggregati di cellule COS in Collagen Matrix <ol><li> Utilizzando un ago di tungsteno appena affilato e un paio di pinze multa (# 5 o # 55), rimuovere il mesoderma che circonda il midollo spinale. </li><li> Taglio parallelo al pianale con l&#39;ago di tungsteno, rimuovere con attenzione il quinto ventrale del midollo spinale. </li><li> Bisecare l&#39;espianto dorsale della colonna vertebrale, assicurandosi che i bordi sono tagliati in modo pulito possibile. Ri-tagliare i bordi, se necessario. Trasferimento ogni espianto dorsale spinale sul pad collagene separati utilizzando una pipetta a bocca dotata di un tubo capillare di vetro tirato a ca. 0,5 millimetri di diametro. </li><li> Tagliare l&#39;aggregato di cellule COS transfettate in quadrati di circa la stessa larghezza del bordo laterale del espianto dorsale della colonna vertebrale. </li><li> Trasferire una di queste piazze sul pad collagene con l&#39;espianto dorsale della colonna vertebrale, utilizzando una pipetta a bocca. </li><li> Con la pipetta bocca, aspirare via il liquido in eccesso con la pipetta bocca. Non più di aspirare. </li><li> Applicare 4μl di collagene innescato sul pad, e, utilizzando l&#39;ago di tungsteno, Spostare il collagene negli espianti senza toccare direttamente il tessuto. Spostando l&#39;ago di tungsteno nel collagene che circonda la espianti, orientare il espianti in modo che l&#39;aggregato cellule COS è adiacente al bordo laterale della espianto dorsale della colonna vertebrale. </li><li> Dopo che il collagene è impostato, ripetere l&#39;applicazione di 4μl di collagene per assicurare che il espianti sono completamente racchiusi in collagene. </li><li> In una cappa colture di tessuti, aggiungere 0,5 ml di Opti-MEM 1xP/S/G + e cultura per 30-40 ore. </li><li> Fix espianti per 45 minuti con 0,5 ml paraformaldeide 4% in 1xPBS, mantenendo la piastra sul ghiaccio. </li><li> Lavare due volte con 0,5 ml di una soluzione di blocco dei 1xPBS + 0,1% Triton-X100 HIGS +1% (PBTN). Blocco a 4 ° C per almeno un paio d&#39;ore, una notte di incubazione è preferibile e quindi elaborare mediante immunoistochimica. </li></ol> Parte 5: Visualizzazione di assoni commissurali di Immunoistochimica <ol><li> Dopo aver bloccato in PBTN, tagliare i espianti dalla piastra ben 4 con una pinza e mettere in un piatto ben 48. </li><li> Aggiungere 200μl di anticorpi primari in ogni pozzetto e lasciare per una notte a 4 ° C. Per visualizzare gli assoni commissurali, utilizzare una soluzione 1:06 di topo anti-Tag1 anticorpi. Per determinare se la transfezione è riuscita, anche un anticorpo primario contro sia la molecola di segnalazione in fase di test, o l&#39;epitopo rilevante se la molecola di segnalazione è stato epitopo tag. </li><li> Lavare ogni campione per 5 x 1 ora in PBTN a 4 ° C. </li><li> Aggiungere 200μl di anticorpo secondario in ogni pozzetto e lasciare per una notte a 4 ° C. Se gli anticorpi anti-Tag1 sono state usate come l&#39;anticorpo primario, utilizzare un anti capra anticorpi IgM secondario del mouse abbinato a uno o FITC Cy3. Se si utilizza la luce anticorpi sensibili secondario accoppiato al FITC o Cy3, tenere il 48 e piatto coperto in fogli da questo punto in poi. </li><li> Lavare ogni campione di 5 x 1 ora ciascuna a PBTN a 4 ° C. </li><li> Trasferimento espianto di un 3-scivolo e depressione, rimuovere il liquido in eccesso, montare in Vectashield medio e coprioggetto. Conservare in una scatola luminosa di sicurezza sia a 4 ° C o -20 ° C. </li></ol> Parte 6: immagini rappresentative di espianti In un esperimento riuscito, l&#39;espianto e aggregati COS rimarrà adiacenti l&#39;uno all&#39;altro e non allontanarsi, come il collagene set. Non ci sarà crescita eccessiva minima degli assoni; significativa crescita eccessiva assonale indica che l&#39;espianto dorsale della colonna vertebrale è stata colta per troppo tempo. Non ci saranno blebs crescente l&#39;espianto; blebbing si verifica se il tessuto viene a contatto diretto con il terreno di coltura. La capacità della molecola di segnalazione di riorientare assoni viene valutato usando la microscopia confocale. Il grado di riorientamento assone può essere quantificata misurando l&#39;angolo medio di riorientamento degli assoni più vicino al aggregato di cellule COS 3. Come mostrato nella Figura 1, le cellule che esprimono COS myc-tag BMP7 (blu) riorientare Tag1 + assoni commissurali (verde) di distanza dalla fonte di BMP7 (Figura 1B) simile al modo in cui un espianto di lamiera del tetto respinge assoni commissurali (Figura 1A). COS cellule che esprimono un vettore di controllo non hanno alcun effetto sull&#39;orientamento degli assoni commissurali 3,4. Questi espianti sono stati etichettati con anticorpi contro il fattore di trascrizione Isl1 / 2 (rosso), che decora i motoneuroni e interneuroni dorsale. Questo risultato è stato il primo segnale che un membro della famiglia della media BMP l&#39;attività dei 3 lamiera del tetto chemorepellent. <img src="/files/ftp_upload/1853/1853fig1tmb.jpg" border="0" /> Figura 1: Si prega di <a href="https://www.jove.com/files/ftp_upload/1853/1853fig1.jpg">cliccare qui</a> per vedere una versione più grande della figura 1.

<ol>
	<li>Altman, J., Bayer, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The development of the rat spinal cord. , (1984).</li><li>Placzek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+recombinations+in+collagen+gels.">Tissue recombinations in collagen gels.</a> Methods in molecular biology. , 325-335 (2008).</li><li>Augsburger, A., Schuchardt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMPs+as+mediators+of+roof+plate+repulsion+of+commissural+neurons.">BMPs as mediators of roof plate repulsion of commissural neurons.</a> Neuron. 24, 127-141 (1999).</li><li>Butler, S. J., Dodd, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+BMP+heterodimers+in+roof+plate-mediated+repulsion+of+commissural+axons.">A role for BMP heterodimers in roof plate-mediated repulsion of commissural axons.</a> Neuron. 38, 389-401 (2003).</li><li>Kennedy, T. E., Serafini, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Netrins+are+diffusible+chemotropic+factors+for+commissural+axons+in+the+embryonic+spinal+cord.">Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord.</a> Cell. 78, 425-435 (1994).</li><li>Charron, F., Stein, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphogen+sonic+hedgehog+is+an+axonal+chemoattractant+that+collaborates+with+netrin-1+in+midline+axon+guidance.">The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance.</a> Cell. 113, 11-23 (2003).</li><li>Lumsden, A. G., Davies, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotropic+effect+of+specific+target+epithelium+in+the+developing+mammalian+nervous+system.">Chemotropic effect of specific target epithelium in the developing mammalian nervous system.</a> Nature. 323, 538-539 (1986).</li><li>Dodd, J., Morton, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+regulation+of+axonal+glycoprotein+expression+on+subsets+of+embryonic+spinal+neurons.">Spatial regulation of axonal glycoprotein expression on subsets of embryonic spinal neurons.</a> Neuron. 1, 105-116 (1988).</li><li>Yamauchi, K., Phan, K. D., Butler, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+type+I+receptor+complexes+have+distinct+activities+mediating+cell+fate+and+axon+guidance+decisions.">BMP type I receptor complexes have distinct activities mediating cell fate and axon guidance decisions.</a> Development. 135, 1119-1128 (2008).</li></ol>

I fattori critici che determinano il successo nello svolgimento di questo saggio sono prima, il tessuto non devono essere trattati con dispasi per un periodo troppo prolungato, tale trattamento sarà seguito nel tessuto diventando molto appiccicosa e con ridotta capacità di sopravvivenza. Due, il collagene deve essere perfettamente innescato e tenuta in ghiaccio il più possibile. Diventerà sempre più difficile da gestire se inizia a impostare, ad esempio si tingono di rosa. Tre, l&#39;ago di tungsteno deve essere se...

assaying the ability of diffusible signaling molecules to reorient embryonic spinal commissural axons

Dorsale commissurali assoni nel midollo spinale dei vertebrati 1 sono stati di un sistema prezioso modello in cui identificare i segnali di guida degli assoni. Qui, descriviamo un test in vitro &quot;, il saggio riorientamento&quot;, che è stato ampiamente utilizzato per studiare l&#39;effetto dei segnali estrinseci ed intrinseci sull&#39;orientamento degli assoni commissurali 2. Questo test è stato sviluppato da numerose persone nei laboratori di Jane Dodd, Thomas Jessell e Andrew Lumsden (si vedano i ringraziamenti per maggiori dettagli) e le versioni di questo test sono stati utilizzati per dimostrare l&#39;attività di riorientamento delle molecole chiave di guida degli assoni, tra cui il chemorepellent in BMP tetto piatto 3,4 e le attività di chemoattractive Netrin1 5 e Sonic hedgehog (Shh) 6 in la lastra di fondo nel midollo spinale. Espianti composto da 2-3 segmenti della dorsale due terzi del midollo spinale vengono sezionati fin dal primo giorno embrionale (E) 11 ratti e coltivate in tre gel collagene dimensionale 7. E11 espianti dorsale spinale contengono neonata commissurali neuroni, che possono essere identificati dal loro espressione della glicoproteina assonale, Tag1 8. Nel corso di 30-40 ore nella cultura, la traiettoria assone commissurali è riassunta in queste espianti dorsale con un decorso simile a quello visto in vivo. Questa traiettoria assonale può essere contestata o posizionando i tessuti di prova o un aggregato di cellule COS esprimono una molecola di segnalazione candidato in contatto con uno dei bordi laterali del espianto dorsale. Assoni commissurali si estende in prossimità del tessuto aggiunto crescerà sotto l&#39;influenza sia della lamiera del tetto endogeno e segnali del tessuto ectopico laterale. Il grado con cui sono riorientato assoni commissurali in queste circostanze può essere quantificato. Da questo test, è possibile sia per esaminare la sufficienza di un segnale particolare di riorientare gli assoni commissurali 3,4 come pure la necessità di questo segnale per dirigere la traiettoria commissurali 9.

Watch this Scientific Journal Video about Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons at JoVE.com

Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

Questo test valuta la capacità di una molecola di segnalazione, qui proteina morfogenetica dell&#39;osso 7 (BMP7), di riorientare gli assoni commissurali. Un espianto di midollo embrionale dorsale spinale è colta adiacente a un aggregato di cellule secernenti COS i fattori di crescita candidato. Riorientato assoni commissurali crescita all&#39;interno del espianto sono visualizzati mediante immunoistochimica.

Capacità di analizzare il diffusibili molecole di segnalazione per l&#39;orientamento embrionali assoni spinali commissurali

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals.  Here, we ...

assaying-ability-diffusible-signaling-molecules-to-reorient-embryonic

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JoVE Journal

Biology

8.7K Views.  University of Southern California. Questo test valuta la capacità di una molecola di segnalazione, qui proteina morfogenetica dell'osso 7 (BMP7), di riorientare gli assoni commissurali. Un espianto di midollo embrionale dorsale spinale è colta adiacente a un aggregato di cellule secernenti COS i fattori di crescita candidato. Riorientato assoni commissurali crescita all'interno del espianto sono visualizzati mediante immunoistochimica.

Video: Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

Fabbricazione di un dispositivo a microfluidi per la compartimentazione dei Neuron Soma e assoni

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

Ricerca

Biologia

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

Spinal Cord Elettrofisiologia

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

Embrionali di topo Cultura Lung, un sistema per valutare i meccanismi molecolari della Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.

In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.

Adenovirus-mediata rimozione genetica di molecole di segnalazione in fibroblasti di topo coltivate primarie embrionali

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP &amp; FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

Fotodecolorazione saggi (FRAP e FLIP) per misurare la dinamica delle proteine ​​della cromatina in Living cellule staminali embrionali

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

Saggiare l&#39;attività chinasi di LRRK2 In vitro</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Un sistema per<em&gt; Ex vivo</emColtura&gt; del pancreas embrionale

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Espianti blastomero al test per l&#39;Impegno cellulare Fate durante lo sviluppo embrionale

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

La stimolazione meccanica indotta calcio Wave Propagation in monostrati cellulari: l&#39;esempio di bovini cellule endoteliali corneali

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).