Dieses Protokoll wurde in den Labors von Jane Dodd, Thomas Jessell und Andrew Lumsden entwickelt. Viele Menschen, darunter Konrad Basler, Ann Calof, Thomas Edlund, Phil Hamilton, Domna Karagogeos, Ariel Ruiz i Altaba und Toshiya Yamada bestimmt, wie die Kultur Explantate des Rückenmarks in Kollagen. Marysia Placzek und Marc Tessier-Lavigne Pionier in der Technik der Verwendung von Axon-Wachstum von in vitro Explantate als Mittel zur Identifizierung von Axon Führung Moleküle. Die Arbeit in der Butler Labor ist durch Zuschüsse der March of Dimes und R01 NS063999 von NIH / NINDS unterstützt.

Teil 1: Vorbereitung der Aggregate von transfizierten COS-Zellen mit hängenden Tropfen <ol><li> Seed COS-7 (COS)-Zellen in 35-mm-Kulturschalen. Als sie 80% Konfluenz, transfizieren 1 g des Expressionsplasmids in die Zellen mit Lipofectamine2000 nach Angaben des Herstellers s-Protokoll zu erreichen. </li><li> Zur Vorbereitung hängenden Tropfen absaugen Transfektionsmedium und spülen Sie die transfizierten COS-Zellen mit 1 ml 1x Phosphat-gepufferte Lösung (PBS). Gönnen Zellen mit 0,5 ml Enzym-freie Zelle Dissoziation Medium für 15 Minuten. 1ml einer Lösung von Opti-MEM + 1x Penicillin / Streptomycin / Glutamin (P / S / G) + 10% fötalem Rinderserum (FBS), um die Reaktion zu stoppen. </li><li> Man reibt die Zellen, um sie von der Oberfläche der Kulturschale zu entfernen und in ein 15 ml konische Röhre. Spin 2 Minuten lang bei 2000K die Zellen zu pelletieren. Überstand entfernen und Pellet in 100 &amp;mgr; l der Opti-MEM + 1x P / S / G + 10% FBS. </li><li> Spot mehrere 20 &amp;mgr; l Tropfen auf die Innenseite einer 35-mm-Kulturschale Deckel, Invertzucker Deckel und am oberen Rand des Boden der Schale und verlassen in einem 37 ° C Inkubator für mehrere Stunden, bis die Zellen aggregieren. </li></ol> Teil 2: Herstellung von Rückenmark Explantate <ol><li> Dissect E11 Ratte Embryonen aus der Gebärmutter der Mutter und halten auf dem Eis in L15-Medium bis es gebraucht wird. </li><li> Mit einem geschärften Wolframnadel, entfernen Sie eine 4-6 Segmentabschnitt aus dem Kofferraum eines jeden Embryo, aus der Region unmittelbar unterhalb forelimb Knospe. Mit einem Kunststoff-Pipette, sammeln die Gewebestücke in eine Vertiefung einer 4-Well Nunc Schüssel und halten Sie auf dem Eis. </li><li> Wenn alle Segmente des Embryos wurden seziert, die Kunststoff-Pipette, um die Stücke in eine Lösung von 1 ml L15 + 1mg Dispase in einem zweiten Brunnen der Nunc Gericht zu übertragen. Inkubieren bei Raumtemperatur nicht länger als 5 Minuten. Nicht zu bebrüten die Gewebestücke mit Dispase. </li><li> Während der Inkubation add 0,5 ml hitzeinaktiviertem normalem Ziegenserum (Treble) zu etwa 10 ml der L15 in eine Petrischale und wirbeln zu mischen. Transfer 1ml dieser Lösung in einen dritten Brunnen der Nunc Schüssel und übertragen Sie die Gewebestücke in diese Lösung, wenn die Inkubationszeit ist vorbei. Keep on ice. Hinweis: Die nachfolgende Dissektion wird erleichtert, wenn die Gewebestücke zu &quot;Ruhe&quot; auf Eis für eine Stunde erlaubt sind. </li></ol> Teil 3: Priming Collagen <ol><li> Add 40 ul 10x Minimal Essential Medium zu 360μl Kollagen, schnell und gründlich zu mischen, indem flicking oder kurz vortexen die Röhre. Spin down schnell in einer picofuge. Die Lösung wird gelb sein. Keep on ice so viel wie möglich. </li><li> Fügen Sie genug 0.8M Natriumhydrogencarbonat (NaHCO 3) auf die Kollagen-Lösung leicht orange drehen (siehe Hinweis unten). Wieder schnell und gründlich zu mischen, indem flicking und Spin-down kurz in einer picofuge. Bleibt die Lösung rosa nach dem Mischen, Sie haben zu viel NaHCO 3 zugegeben und müssen wieder von vorne anfangen mit ein frisches Röhrchen aus Kollagen. </li><li> An diesem Punkt der Kollagen-Lösung ist grundiert. Es wird flüssig bleiben auf dem Eis, sondern wird innerhalb von ca. 5 Minuten (Drehen pink) verfestigen, wenn auf Raumtemperatur gebracht. </li><li> Spot 20 &amp;mgr; l von Kollagen in jede Vertiefung einer 4-Well Nunc Gericht. Mit der Spitze des Pipette verteilt das Kollagen zu einem kleinen &quot;pad&quot; zu bilden. Lassen Sie das Kollagen bei Raumtemperatur eingestellt. </li></ol> Hinweis: Die genaue Höhe der NaHCO 3 muss für jede Charge von Kollagen titriert werden. Starten niedrig (11μl) und fügen Sie dann 0,5-1μl schrittweise, bis die Lösung eine leichte Schatten orange. Die &quot;richtige&quot; Menge ist in der Regel 1UL weniger als die kleinste Menge an NaHCO 3, dass das Kollagen rosa wiederum würde. Die Höhe des NaHCO 3 nicht linear nach oben oder unten skalieren. Teil 4: Präparation Rückenmark Explantate und Positionierung von ihnen mit COS Zellaggregate in Kollagen-Matrix <ol><li> Mit einem frisch geschärften Wolframnadel und ein Paar von feinen Pinzette (# 5 oder # 55), entfernen Sie das Mesoderm Umgebung des Rückenmarks. </li><li> Schneiden parallel zur Bodenplatte mit dem Wolfram-Nadel, entfernen Sie vorsichtig den ventralen Fünftel des Rückenmarks. </li><li> Halbierung der dorsalen Wirbelsäule Explantation, um sicherzustellen, dass Kanten so sauber wie möglich schneiden. Re-Trim die Kanten, wenn nötig. Übertragen Sie jede dorsalen Explantat auf separate Kollagen-Pads mit einem Mund pipettieren mit einer Glaskapillare gezogen, um ca. ausgestattet. 0,5 mm im Durchmesser. </li><li> Schneiden Sie die Summe von transfizierten COS-Zellen in Quadrate von ungefähr die gleiche Breite wie die seitlichen Rand des dorsalen Explantation. </li><li> Übertragen eines dieser Quadrate auf die Kollagen-Pad mit der dorsalen Wirbelsäule Explantation, mit einem Mund pipettieren. </li><li> Mit dem Mund pipettieren, absaugen überschüssige Flüssigkeit mit dem Mund pipettieren. Nicht zu saugen. </li><li> Bewerben 4μl von grundierten Kollagen auf dem Pad, und mit dem Wolfram-NadelBewegen sich die Kollagen über die Explantate ohne direkte Berührung des Gewebes. Durch Bewegen der Wolframnadel in das Kollagen um die Explantate, orientieren die Explantate, so dass die COS-Zellen aggregieren neben dem seitlichen Rand der dorsalen Explantat wird. </li><li> Nach dem Kollagen gesetzt hat, wiederholen Sie die Anwendung von 4μl von Kollagen, um sicherzustellen, dass die Explantate komplett eingehüllt in Kollagen sind. </li><li> In einer Gewebekultur Kapuze, fügen Sie 0,5 ml Opti-MEM + 1xP/S/G und Kultur für 30-40 Stunden. </li><li> Fix Explantate für 45 Minuten mit 0,5 ml 4% Paraformaldehyd in 1xPBS, halten die Platte auf Eis. </li><li> Zweimal waschen mit 0,5 ml eines blockierenden Lösung von 1xPBS + 0,1% Triton-X100 +1% HIGS (PBTN). Block bei 4 ° C für mindestens ein paar Stunden, ist eine Inkubation über Nacht vorzuziehen und dann verarbeiten durch Immunhistochemie. </li></ol> Teil 5: Visualisierung von kommissuralen Axone durch Immunhistochemie <ol><li> Nach der Blockierung in PBTN, schneiden Sie die Explantate aus der 4-Well-Platte mit einer Pinzette und in eine 48-Well-Schale. </li><li> Add 200 ul der primäre Antikörper in jede Vertiefung und lassen Sie über Nacht bei 4 ° C. Zur Visualisierung kommissuralen Axone, verwenden Sie einem 1:6-Lösung von Maus-anti-Tag1-Antikörper. Um festzustellen, ob die Transfektion erfolgreich war, auch einen primären Antikörper entweder gegen das Signalmolekül getestet, oder der jeweilige Epitop, wenn das Signalmolekül hat Epitop markierten worden. </li><li> Wash jede Probe für 5 x 1 Stunde in PBTN bei 4 ° C. </li><li> Add 200 ul des sekundären Antikörper in jede Vertiefung und lassen Sie über Nacht bei 4 ° C. Wenn Anti-Tag1 Antikörper als primärer Antikörper verwendet wurden, verwenden Sie eine Ziege anti-Maus IgM sekundäre Antikörper gekoppelt, um entweder FITC oder Cy3. Bei Verwendung von lichtempfindlichen Sekundärantikörper FITC oder Cy3 gekoppelt, halten Sie die 48-well Schale in Folie ab diesem Zeitpunkt erfasst. </li><li> Wash jeder Probe 5 x je 1 Stunde in PBTN bei 4 ° C. </li><li> Transfer Explantation zu einem 3-gut Depression rutschen, zu entfernen überschüssige Flüssigkeit, mount in Vectashield mittel-und Deckglas. An einem leichten Safe entweder bei 4 ° C oder -20 ° C. </li></ol> Teil 6: Repräsentative Bilder von Explantaten In einem erfolgreichen Experiment, wird das Explantat und COS Aggregat bleiben nebeneinander und nicht auseinander driften, wie die Kollagen-Sets. Es wird minimal Überwucherung der Axone werden; signifikante axonale Überwucherung zeigt, dass die dorsalen Explantation zu lange war, kultiviert. Es wird keine Bläschen wächst aus dem Explantat werden; blebbing tritt auf, wenn das Gewebe in direkten Kontakt mit dem Kulturmedium. Die Fähigkeit der Signalmolekül auf Axone Neuausrichtung wird anhand der konfokalen Mikroskopie. Das Ausmaß der Axon Neuorientierung kann durch Messung der mittleren Winkel der Neuorientierung der Axone am nächsten an der COS-Zelle Aggregat 3 quantifiziert werden. Wie in Abbildung 1 dargestellt, zum Ausdruck COS-Zellen myc-markierten BMP7 (blau) neu zu orientieren Tag1 + kommissuralen Axone (grün) entfernt von der Quelle des BMP7 (Abbildung 1B), ähnlich der Art und Weise, auf die ein Explantat der Dachplatte abstößt kommissuralen Axone (Abb. 1A). COS-Zellen, die eine Steuerung Vektor haben keinen Einfluss auf die Ausrichtung der kommissuralen Axone 3,4. Diese Explantate wurden auch mit Antikörpern gegen den Transkriptionsfaktor Isl1 / 2 (rot), die Motoneuronen und dorsalen Interneuronen schmückt gekennzeichnet. Dieses Ergebnis war der erste Hinweis darauf, dass ein Mitglied der BMP-Familie vermittelt die Aktivität der Dachplatte chemorepellent 3. <img src="/files/ftp_upload/1853/1853fig1tmb.jpg" border="0" /> Abbildung 1: Bitte <a href="https://www.jove.com/files/ftp_upload/1853/1853fig1.jpg">klicken Sie hier,</a> um eine größere Version der Abbildung 1 zu sehen.

<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>

Die kritischen Faktoren, die den Erfolg bei der Durchführung dieses Tests bestimmen, sind erstens die Gewebe nicht mit Dispase für zu lange einen Zeitraum behandelt werden, wird eine solche Behandlung in das Gewebe zu sehr klebrig und mit verminderter Tragfähigkeit führen. Zwei, muss das Kollagen perfekt vorbereitet werden und auf Eis gehalten so viel wie möglich. Es wird zunehmend schwieriger zu handhaben, wenn sie gesetzt beginnt, dh sich rosa färben. Drei, muss der Wolfram-Nadel stets sehr scharf sein.

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

Dorsal kommissuralen Axone in der Wirbeltier-Rückenmark 1 haben einen unschätzbaren Modell, in dem Axon Führung Signale zu identifizieren gewesen. Hier beschreiben wir ein In-vitro-Assay, &quot;die Neuausrichtung assay&quot;, die verwendet wurde ausgiebig um die Wirkung von äußeren und inneren Signale von der Orientierung der Axone kommissuralen-2-Studie. Dieser Test wurde von zahlreichen Menschen in den Labors von Jane Dodd, Thomas Jessell und Andrew Lumsden entwickelt (siehe Anerkennungen für weitere Details) und Versionen dieses Tests wurden verwendet, um die Neuausrichtung Aktivitäten der Schlüssel Axon Führung Moleküle nachzuweisen, einschließlich der BMP chemorepellent in der Dachblech 3,4 und die chemoattractive Aktivitäten Netrin1 5 und Sonic Hedgehog (SHH) 6 in die Bodenplatte in das Rückenmark. Explantate aus 2-3 Segmenten der dorsalen zwei Drittel des Rückenmarks sind aus embryonalen Tag (E) 11 Ratten seziert und kultiviert im dreidimensionalen Kollagen-Gelen 7. E11 dorsalen Explantate enthalten neu geboren kommissuralen Neuronen, die durch ihre axonalen Ausdruck des Glykoprotein, Tag1 8 identifiziert werden können. Im Laufe von 30-40 Stunden in Kultur, ist die kommissuralen Axon Flugbahn in diesen dorsalen Explantate mit einer zeitlichen Verlauf ähnlich dem in vivo gesehen rekapituliert. Diese Axone Flugbahn kann, indem entweder Test Gewebe oder eine COS-Zelle aggregieren Ausdruck eines Kandidaten Signalmolekül in Kontakt mit einem der seitlichen Ränder der dorsalen Explantation angefochten werden. Kommissuralen Axone, die sich in der Nähe der beigefügten Gewebe wird unter dem Einfluss der beiden endogenen Dachblech und Signale aus der ektopischen seitlichen Gewebe wachsen. Der Grad der kommissuralen Axone unter diesen Umständen neu ausgerichtet werden kann quantifiziert werden. Unter Verwendung dieses Tests ist es möglich, sowohl auf die Angemessenheit eines bestimmten Signals, um neu zu orientieren kommissuralen Axone 3,4 sowie die Notwendigkeit für dieses Signal an die kommissuralen Flugbahn 9 direkte untersuchen.

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

Dieser Test bewertet die Fähigkeit eines Signalmolekül, hier Bone Morphogenetic Protein 7 (BMP7), um kommissuralen Axone neu zu orientieren. Eine Explantation von embryonalen Rückenmark kultiviert wird neben einem Aggregat von COS-Zellen sekretieren der Kandidat Wachstumsfaktoren. Umorientiert kommissuralen Axone wachsen innerhalb der Explantation werden visualisiert durch Immunhistochemie.

Testen der Fähigkeit des diffusiblen Signalmoleküle zu embryonalen Spinal kommissuralen Axone neu ausrichten

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

Research

JoVE Journal

Biology

8.7K Aufrufe.  University of Southern California. Dieser Test bewertet die Fähigkeit eines Signalmolekül, hier Bone Morphogenetic Protein 7 (BMP7), um kommissuralen Axone neu zu orientieren. Eine Explantation von embryonalen Rückenmark kultiviert wird neben einem Aggregat von COS-Zellen sekretieren der Kandidat Wachstumsfaktoren. Umorientiert kommissuralen Axone wachsen innerhalb der Explantation werden visualisiert durch Immunhistochemie.

Serie: 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.

Herstellung eines mikrofluidischen Vorrichtung für die Abschottung der Neuron Soma und Axonen

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

Forschung

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 Elektrophysiologie

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.

Embryonalen Maus-Lung Kultur, A-System, die molekularen Mechanismen des Branching Bewerten

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-vermittelte genetische Entfernung von Signalmolekülen in kultivierten primären embryonalen Maus-Fibroblasten

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 Assays (FRAP &amp; FLIP) zu Chromatin Protein Dynamics in Living embryonalen Stammzellen Measure

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.

Untersuchen der Kinase-Aktivität von 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.

Ein System für<em&gt; Ex vivo</em&gt; Kultivierung von embryonalen Pankreas

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.

Blastomere Explantate für das Schicksal der Zelle Engagement während der embryonalen Entwicklung Testen

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.

Mechanische Stimulation induzierte Calcium Wellenausbreitung in Zellmonoschichten: Das Beispiel Bovine Endothelzellen

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).