Ce protocole a été développé dans les laboratoires de Jane Dodd, Thomas et Andrew Lumsden Jessell. Beaucoup de gens, y compris Konrad Basler, Ann Calof, Thomas Edlund, Phil Hamilton, Domna Karagogeos, Ariel Ruiz i Altaba et Toshiya Yamada déterminé comment la culture des explants de moelle épinière dans le collagène. Marysia Placzek et Marc Tessier-Lavigne pionnier de la technique d&#39;utilisation de la croissance axonale à partir dans des explants in vitro comme un moyen d&#39;identifier les molécules de guidage axonal. Le travail dans le laboratoire de Butler est soutenu par des subventions de la Mars of Dimes et R01 NS063999 du NIH / NINDS.

Partie 1: Préparation des agrégats de cellules COS transfectées avec gouttes suspendues <ol><li> COS-7 de semences (COS) des cellules dans des boîtes de culture de 35 mm. Quand ils atteignent 80% de confluence, 1 pg transfecter du plasmide d&#39;expression dans les cellules en utilisant Lipofectamine2000 selon le protocole du fabricant. </li><li> Pour préparer des gouttes pendantes, aspirer le milieu de transfection et rincer les cellules transfectées COS avec 1ml de solution tamponnée de phosphate 1x (PBS). Traiter les cellules avec 0,5 ml d&#39;enzyme sans dissociation cellulaire moyen pendant 15 minutes. Ajouter 1 ml d&#39;une solution d&#39;Opti-MEM + 1x pénicilline / streptomycine / glutamine (P / S / G) + sérum fœtal bovin 10% (FBS) pour arrêter la réaction. </li><li> Triturer les cellules pour les enlever de la surface de la boîte de culture et de transférer dans un tube 15ml conique. Spin 2 minutes à 2000K pour culotter les cellules. Retirer le surnageant et remettre le culot dans 100 ul d&#39;Opti-MEM + 1x P / S / G de FBS + 10%. </li><li> Spot de plusieurs gouttes de 20 pi à l&#39;intérieur d&#39;un couvercle de boîte de culture 35mm, inverser le couvercle et placez-le sur le fond du plat et laisser dans un incubateur à 37 ° C pendant plusieurs heures jusqu&#39;à ce que les cellules agrégées. </li></ol> Partie 2: Préparation des explants dorsaux de la moelle épinière <ol><li> Disséquer embryons de rat E11 de l&#39;utérus de la mère et de garder sur la glace dans du milieu L15 jusqu&#39;à ce que nécessaire. </li><li> Avec une aiguille de tungstène aiguisé, enlever un segment de l&#39;article 4-6 du tronc de chaque embryon, à partir de la région immédiatement en dessous de patte avant bourgeon. Avec une pipette en plastique, de recueillir les morceaux de tissus dans un puits d&#39;une antenne 4-même et de garder Nunc sur la glace. </li><li> Lorsque tous les segments d&#39;embryons ont été disséqués, utilisez la pipette en plastique pour transférer les morceaux dans une solution de 1ml L15 + 1mg dispase dans un deuxième puits de la vaisselle Nunc. Incuber à température ambiante pendant plus de 5 minutes. Ne pas trop incuber les morceaux de tissu avec de la dispase. </li><li> Durant l&#39;incubation, ajouter 0,5 ml de la chaleur du sérum de chèvre inactivé normal (HIGS) à environ 10ml de L15 dans une boîte de Pétri et agiter pour mélanger. Transfert 1ml de cette solution dans un troisième puits du plat Nunc et de transférer les morceaux de tissu dans cette solution lorsque l&#39;incubation est terminée. Restez sur la glace. Remarque: la dissection subséquente sera plus facile si les morceaux de tissu sont autorisés à «reposer» sur la glace pendant une heure. </li></ol> Partie 3: Collagène Amorçage <ol><li> Ajouter 40μl 10x milieu minimal essentiel au collagène 360μl, mélanger rapidement et complètement par agitation ou brièvement le tube de vortex. Isoler rapidement dans un picofuge. La solution sera jaune. Gardez sur la glace autant que possible. </li><li> Ajouter suffisamment bicarbonate de sodium 0,8 M (NaHCO 3) pour mettre la solution de collagène légèrement orangé (voir note ci-dessous). Encore une fois, mélanger rapidement et complètement en feuilletant, et centrifuger brièvement dans un picofuge. Si la solution reste rose après mélange, vous avez ajouté trop de NaHCO 3 et devrez recommencer avec un nouveau tube de collagène. </li><li> A ce point la solution de collagène est amorcée. Il restera liquide sur la glace, mais va se solidifier dans les 5 minutes (en tournant rose) lorsqu&#39;elle est amenée à température ambiante. </li><li> 20 pi spot de collagène dans chaque puits d&#39;un plat 4-même Nunc. En utilisant la pointe de votre pipette, étalé le collagène pour former un petit &quot;pavé&quot;. Laissez le collagène mis à température ambiante. </li></ol> Remarque: Le montant exact de NaHCO 3 devront être titré pour chaque lot de collagène. Démarrer faible (11μl), puis ajouter 0,5 1 microlitre par étapes jusqu&#39;à ce que la solution est une légère teinte d&#39;orange. La &quot;bonne&quot; quantité est généralement inférieure à la 1UL plus petite quantité de NaHCO 3, que ferait le rose de collagène. Le montant de NaHCO 3 n&#39;a pas d&#39;échelle linéaire vers le haut ou vers le bas. Partie 4: Dissection d&#39;explants dorsaux de la moelle épinière et le positionnement d&#39;entre eux avec des agrégats de cellules COS matrice collagène <ol><li> En utilisant une aiguille de tungstène fraîchement aiguisée et une paire de pinces fines (# 5 ou # 55), retirez le mésoderme qui entoure la moelle épinière. </li><li> Coupe parallèle à la plaque au sol avec l&#39;aiguille de tungstène, retirez soigneusement le cinquième ventrale de la moelle épinière. </li><li> Couper en deux l&#39;explant épinière dorsale, en s&#39;assurant que les bords sont aussi proprement que possible coupées. Re-découpez les bords, si nécessaire. Transfert chaque explant épinière dorsale sur coussinets collagène séparées utilisant une pipette bouche équipé d&#39;un tube capillaire en verre tiré à env. 0,5 mm de diamètre. </li><li> Couper l&#39;agrégat de cellules COS transfectées en carrés d&#39;environ la même largeur que le bord latéral de l&#39;explant épinière dorsale. </li><li> Transfert un de ces carrés sur le tampon de collagène avec l&#39;explant épinière dorsale, à l&#39;aide d&#39;une pipette bouche. </li><li> Avec la pipette la bouche, aspirer hors tout excès de liquide à l&#39;aide de la pipette bouche. Ne pas trop aspirer. </li><li> Appliquer 4μl de collagène amorcée sur le pavé,et, en utilisant l&#39;aiguille de tungstène, déplacez le collagène au cours de la explants, sans toucher directement les tissus. En déplaçant l&#39;aiguille de tungstène dans le collagène entourant les explants, orienter les explants de sorte que l&#39;ensemble des cellules COS est adjacent au bord latéral de l&#39;explant épinière dorsale. </li><li> Après le collagène a mis, répéter l&#39;application de 4μl de collagène afin de s&#39;assurer que les explants sont entièrement revêtus de collagène. </li><li> Dans une hotte de culture de tissus, ajouter 0,5 ml d&#39;Opti-MEM 1xP/S/G + et la culture pour 30-40 heures. </li><li> Fix explants pendant 45 minutes avec du paraformaldéhyde 4% dans 0,5 ml 1xPBS, en gardant la plaque sur la glace. </li><li> Laver deux fois avec 0,5 ml d&#39;une solution de blocage des 1xPBS + 0,1% de Triton-X100 HIGS 1% (PBTN). Bloc à 4 ° C pendant au moins une couple d&#39;heures, une nuit d&#39;incubation est préférable et ensuite traiter par immunohistochimie. </li></ol> Partie 5: Visualisation des axones commissuraux par immunohistochimie <ol><li> Après le blocage de PBTN, couper les explants de la plaque 4 et avec une pince et le placer dans un plat de 48 puits. </li><li> Ajouter 200 ul d&#39;anticorps primaires dans chaque puits et laisser une nuit à 4 ° C. Pour visualiser les axones commissuraux, utiliser une solution 1:6 de souris anti-Tag1 anticorps. Pour déterminer si la transfection a été un succès, incluent également un anticorps primaire contre la molécule de signalisation soit mis à l&#39;essai ou l&#39;épitope pertinente si la molécule de signalisation a été épitope étiqueté. </li><li> Lavez chaque échantillon de 5 x 1 heure dans PBTN à 4 ° C. </li><li> Ajouter 200 ul d&#39;anticorps secondaire dans chaque puits et laisser une nuit à 4 ° C. Si l&#39;anti-Tag1 anticorps ont été utilisés comme anticorps primaire, utiliser un anticorps de chèvre anti IgM de souris secondaire couplé à la FITC ou soit Cy3. Si vous utilisez sensible à la lumière des anticorps secondaire couplé à la FITC ou Cy3, garder le plat de 48 puits couvert de feuilles à partir de ce point. </li><li> Laver chaque échantillon de 5 x 1 heure chacune dans PBTN à 4 ° C. </li><li> Transfert explant à une diapositive de dépression 3-même, enlever l&#39;excès de liquide, monter dans Vectashield moyen et lamelle. Conserver dans une boîte de lumière sécuritaire soit à 4 ° C ou -20 ° C. </li></ol> Partie 6: des images représentatives de explants Dans une expérience réussie, l&#39;explant et globale COS restera adjacents les uns aux autres et ne pas dériver à part comme le collagène ensembles. Il y aura une prolifération minimale des axones; significative la prolifération axonale indique que l&#39;explant épinière dorsale a été cultivé pendant trop longtemps. Il n&#39;y aura pas de croissance des bulles de l&#39;explant; blebbing se produit si le tissu est en contact direct avec le milieu de culture. La capacité de la molécule de signalisation de réorienter les axones est évaluée en utilisant la microscopie confocale. L&#39;étendue de la réorientation axonale peut être quantifiée en mesurant l&#39;angle moyen de réorientation des axones plus proche de l&#39;agrégat de cellules COS 3. Comme le montre la figure 1, les cellules COS exprimant myc-taggés BMP7 (bleu) + réorienter Tag1 axones commissuraux (vert) loin de la source de BMP7 (figure 1B), similaire à la manière dont un explant de la plaque de toit repousse des axones commissuraux (figure 1A). Des cellules COS exprimant un vecteur de contrôle n&#39;ont aucun effet sur ​​l&#39;orientation des axones commissuraux 3,4. Ces explants ont également été marquées avec des anticorps contre le facteur de transcription ISL1 / 2 (rouge), qui orne les motoneurones et les interneurones dorsale. Ce résultat a été la première indication que membre de la famille BMP médiateur de l&#39;activité des trois plaque de toit chemorepellent. Figure 1: <a href="https://www.jove.com/files/ftp_upload/1853/1853fig1.jpg"><img src="/files/ftp_upload/1853/1853fig1tmb.jpg" border="0" /></a>

<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>

Les facteurs critiques qui déterminent le succès dans l&#39;accomplissement de ce test sont tout d&#39;abord, le tissu ne doit pas être traitée avec dispase trop prolongée d&#39;une période, un tel traitement se traduira dans le tissu devient très collant et ayant diminué la viabilité. Deux, le collagène doit être parfaitement apprêté et conservés sur la glace autant que possible. Il deviendra plus difficile à gérer si elle commence à mettre, c&#39;est à dire virer au rose. Trois, l&#39;aiguille de tun...

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

Dorsale axones commissuraux de la moelle épinière des vertébrés 1 ont été une précieuse système modèle dans lequel d&#39;identifier des signaux de guidage axonal. Ici, nous décrivons un test in vitro, «le test de réorientation», qui a été largement utilisée pour étudier l&#39;effet des signaux extrinsèques et intrinsèques de l&#39;orientation des axones commissuraux 2. Ce test a été développé par de nombreuses personnes dans les laboratoires de Jane Dodd, Thomas et Andrew Lumsden Jessell (voir les remerciements pour plus de détails) et les versions de cet essai ont été utilisés pour démontrer les activités réorientation des principales molécules de guidage axonal, y compris le chemorepellent dans le BMP 3,4 toit en tôle et les activités des chimioattractives Netrin1 5 et Sonic Hedgehog (Shh) 6 dans la plaque de sol dans la moelle épinière. Explants comprenant 2-3 segments de la dorsale des deux tiers de la moelle épinière sont disséqués par jour embryonnaire (E) 11 rats et de culture en trois dimensions des gels de collagène 7. E11 dorsale explants épinière contiennent les neurones nouvellement né commissurale, qui peuvent être identifiés par leur expression axonale de la glycoprotéine, Tag1 8. Au cours de 30-40 heures de culture, de la trajectoire des axones commissuraux est résumée dans ces explants dorsal avec un cours à temps similaire à celle observée in vivo. Cette trajectoire axonale peut être contestée en plaçant les tissus essai ou un agrégat de cellules COS exprimant une molécule candidate de signalisation en contact avec l&#39;un des bords latéraux de l&#39;explant dorsale. Axones commissuraux s&#39;étendant au voisinage du tissu annexé feront croître sous l&#39;influence de l&#39;ensemble de la plaque de toit endogènes et les signaux provenant du tissu ectopique latéral. Le degré auquel les axones commissuraux sont réorientés dans ces circonstances peuvent être quantifiés. L&#39;utilisation de ce test, il est possible à la fois d&#39;examiner la suffisance d&#39;un signal particulier de réorienter les axones commissuraux 3,4 ainsi la nécessité pour ce signal pour diriger la trajectoire commissurale 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

Ce test évalue la capacité d&#39;une molécule de signalisation, voici protéine morphogénétique osseuse 7 (BMP-7), à réorienter les axones commissuraux. Un explant de la moelle épinière embryonnaire dorsal est cultivée adjacente à un agrégat de cellules COS sécrétant des facteurs de croissance candidat. Réorienté axones commissuraux croissante au sein de l&#39;explant sont visualisées par immunohistochimie.

Dosage de la capacité des molécules de signalisation diffusibles à réorienter embryonnaires épinière axones commissuraux

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

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8.8K vues.  University of Southern California. Ce test évalue la capacité d'une molécule de signalisation, voici protéine morphogénétique osseuse 7 (BMP-7), à réorienter les axones commissuraux. Un explant de la moelle épinière embryonnaire dorsal est cultivée adjacente à un agrégat de cellules COS sécrétant des facteurs de croissance candidat. Réorienté axones commissuraux croissante au sein de l'explant sont visualisées par immunohistochimie.

Vidéo: 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.

Fabrication d&#39;un dispositif microfluidique pour la compartimentation du Neuron Soma et des axones

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

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Biologie

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 électrophysiologie

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.

Culture poumon embryonnaire de la souris, un système pour évaluer les mécanismes moléculaires des 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.

Adénovirus suppression génétique des molécules de signalisation dans les cultures de fibroblastes de souris embryonnaires primaires

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.

Photobleaching essais (FRAP et FLIP) pour mesurer la dynamique des protéines de la chromatine dans Vivre cellules souches embryonnaires

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.

Dosage de l&#39;activité kinase de 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 système d&#39;<em&gt; Ex vivo</emLa culture&gt; de pancréas embryonnaire

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.

Les explants blastomères à l&#39;essai pour l&#39;engagement du destin cellulaire au cours du développement embryonnaire

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 stimulation mécanique induite par le calcium propagation des ondes dans des monocouches de cellules: l&#39;exemple de bovins cellules endothéliales de la cornée

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).

La cartographie du génome à l&#39;échelle du médicament-ADN interactions dans les cellules avec COSMIC (réticulation de petites molécules pour isoler la chromatine)