Este protocolo fue desarrollado en los laboratorios de Jane Dodd, Jessell Thomas y Andrew Lumsden. Muchas personas, incluyendo Konrad Basler, Calof Ann, Edlund Thomas, Hamilton Phil, Karagogeos Domna, Ariel Ruiz i Altaba y Yamada Toshiya determina la forma de explantes de la cultura de la médula espinal en el colágeno. Marysia Placzek y Marc Tessier-Lavigne fue pionero en la técnica de utilización de crecimiento de los axones de in vitro de explantos, como medio de identificación de las moléculas de orientación axón. El trabajo en el laboratorio de Butler es apoyado por becas de la Fundación March of Dimes y NS063999 R01 de NIH / NINDS.

Parte 1: Preparación de los agregados de células transfectadas COS el uso de gotas colgantes <ol><li> COS-7 semillas (COS), las células en placas de cultivo de 35 mm. Cuando llegan a un 80% 1 g confluencia, transfectar del plásmido de expresión en las células utilizando Lipofectamine2000 según el protocolo del fabricante s. </li><li> Para preparar las gotas colgantes, aspirar el medio de transfección y aclarar las células COS transfectadas con 1 ml de solución tampón fosfato 1x (PBS). Tratar las células con 0,5 ml de enzima libre de medio de disociación celular durante 15 minutos. Añadir 1 ml de una solución de Opti-MEM + 1x penicilina / estreptomicina / glutamina (P / S / G) + 10% de suero fetal bovino (SFB) para detener la reacción. </li><li> Triturar las células para eliminar de la superficie de la placa de cultivo y la transferencia en un tubo cónico de 15 ml. Girar durante 2 minutos a 2000K para precipitar las células. Aspirar el sobrenadante y el pellet se resuspende en 100μl de Opti-MEM + 1x P / S / G + 10% de SFB. </li><li> Lugar varias gotas de 20μl en el interior de la tapa de la cultura plato de 35 mm, invertir tapa y coloque en la parte superior de la parte inferior del plato y dejar en una incubadora a 37 ° C durante varias horas hasta que las células agregado. </li></ol> Parte 2: Preparación de explantes dorsal de la médula espinal <ol><li> Diseccionar embriones E11 rata del útero de la madre y mantener en hielo en medio L15 hasta que sea necesario. </li><li> Con una aguja de tungsteno afilado, quitar una sección dedicada al segmento 4.6 a partir del tronco de cada embrión, de la región inmediatamente por debajo de extremidad anterior brote. Con una pipeta de plástico, recoger los trozos de tejido en un pocillo de una placa Nunc de 4 bien y mantener en hielo. </li><li> Cuando todos los segmentos del embrión han sido disecados, el uso de la pipeta de plástico para la transferencia de las piezas en una solución de 1 ml L15 + 1mg dispasa en un segundo pozo de la placa Nunc. Incubar a temperatura ambiente durante no más de 5 minutos. No más de incubar los trozos de tejido con dispasa. </li><li> Durante la incubación, añadir 0,5 ml de suero inactivado por calor normal de cabra (HIGS) a unos 10 ml de L15 en una placa de Petri y agitar para mezclar. Transferencia de 1 ml de esta solución en un tercer pozo de la placa Nunc y la transferencia de las piezas de tejido en esta solución cuando la incubación se ha terminado. Mantener en hielo. Nota: la disección posterior será más fácil si los trozos de tejido se les permite &quot;descansar&quot; en el hielo durante una hora. </li></ol> Parte 3: El colágeno cebado <ol><li> Añadir 40μl 10x Medio Esencial Mínimo de colágeno 360μl, mezcla rápida y completamente por el parpadeo o vortex brevemente el tubo. Girar rápidamente en un picofuge. La solución será de color amarillo. Mantener en hielo tanto como sea posible. </li><li> Agregue bastante bicarbonato de sodio 0,8 M (NaHCO 3) a su vez, la solución de colágeno ligeramente anaranjado (ver nota abajo). Una vez más, mezcla rápida y completamente por el parpadeo, y de girar brevemente en un picofuge. Si la solución permanece rosa después de la mezcla, que ha añadido demasiado NaHCO 3 y tendrá que empezar de nuevo con un nuevo tubo de colágeno. </li><li> En este punto la solución de colágeno está preparado. Seguirá siendo líquido en hielo, pero se solidifica dentro de unos 5 minutos (convirtiendo de color rosa), cuando a temperatura ambiente. </li><li> 20μl punto de colágeno en cada pocillo de una placa Nunc de 4 pocillos. Con la punta de la pipeta, se extendió a cabo el colágeno para formar un pequeño &quot;pad&quot;. Deje que el conjunto de colágeno a temperatura ambiente. </li></ol> Nota: La cantidad exacta de NaHCO 3 tendrán que ser ajustadas individualmente para cada lote de colágeno. Comenzar con dosis bajas (11μl) y luego añadir 0,5 1μl paso a paso hasta que la solución es un leve matiz de color naranja. La cantidad &quot;correcta&quot; es por lo general menos de 1 ul la menor cantidad de NaHCO 3 que a su vez, la rosa de colágeno. La cantidad de NaHCO 3 no se escala linealmente hacia arriba o hacia abajo. Parte 4: La disección de la médula espinal dorsal explantes y Posicionamiento de ellos con los agregados de células COS en la matriz de colágeno <ol><li> Utilizando una aguja de tungsteno y recién afilado y un par de pinzas finas (# 5 y # 55), retire el mesodermo que rodea la médula espinal. </li><li> Corte paralelo a la placa de suelo con la aguja de tungsteno, retire con cuidado la quinta ventral de la médula espinal. </li><li> Atraviesan el explante espinal dorsal, asegurándose de que los bordes son de corte limpio como sea posible. Volver a recortar los bordes, si es necesario. La transferencia de cada explante espinal dorsal en las almohadillas de colágeno por separado con una pipeta de boca provisto de un tubo capilar de vidrio tirado a aprox. 0,5 mm de diámetro. </li><li> Cortar el agregado de células COS transfectadas en cuadrados de aproximadamente el mismo ancho que el borde lateral de la columna dorsal explante. </li><li> La transferencia de uno de estos cuadrados en la plataforma de colágeno con el explante de la columna dorsal, utilizando una pipeta de boca. </li><li> Con la pipeta de la boca, aspirar el exceso de líquido mediante la pipeta de boca. No más de aspirado. </li><li> Aplicar 4μl de colágeno preparados a la almohadilla, y, con la aguja de tungsteno, Mueva el colágeno en los explantes, sin contacto directo con el tejido. Al mover la aguja de tungsteno en el colágeno alrededor de los explantes, orientar a los explantes de modo que la suma células COS se encuentra junto al borde lateral de la columna dorsal explante. </li><li> Después de que el colágeno se ha puesto, repetir la aplicación de 4μl de colágeno para asegurar que los explantes son completamente encerrado en el colágeno. </li><li> En una campana de cultivo de tejidos, añadir 0,5 ml de Opti-MEM 1xP/S/G + y la cultura de 30-40 horas. </li><li> Fix explantes durante 45 minutos con 0,5 ml de paraformaldehído al 4% en 1XPBS, manteniendo la placa en el hielo. </li><li> Lavar dos veces con 0,5 ml de una solución de bloqueo de 1XPBS + 0,1% Tritón-X100 HIGS 1% (PBTN). Bloquear a 4 ° C durante al menos un par de horas, una noche de incubación es preferible y proceso mediante técnicas de inmunohistoquímica. </li></ol> Parte 5: Visualización de los axones de la comisura de inmunohistoquímica <ol><li> Tras el bloqueo en PBTN, cortar los explantes de la placa de 4 y con unas pinzas y colocar en un plato de 48 pocillos. </li><li> Agregar 200μl de los anticuerpos primarios a cada pocillo y dejar toda la noche a 4 ° C. Para visualizar axones de la comisura, use una solución de 1:6 de ratón anti-Tag1 anticuerpos. Para determinar si la transfección fue un éxito, también incluyen un anticuerpo primario contra la molécula de señalización que se prueba, o el epítopo correspondiente, si la molécula de señalización ha sido epítopo etiquetados. </li><li> Lave cada muestra de 5 x 1 hora en PBTN a 4 ° C. </li><li> Agregar 200μl de secundaria de anticuerpos a cada pocillo y dejar toda la noche a 4 ° C. Si el anti-Tag1 anticuerpos fueron utilizados como anticuerpos primarios, use una cabra anti IgM secundario del ratón junto a cualquiera de FITC o Cy3. Si se utiliza la luz secundaria de anticuerpos sensibles acoplado a FITC o Cy3, mantener el plato de 48 bien cubierto en papel de aluminio a partir de ahora. </li><li> Lavar cada muestra de 5 x 1 hora cada uno en PBTN a 4 ° C. </li><li> Transferencia de explante a una diapositiva depresión 3-bien, retirar el exceso de líquido, montaje en Vectashield medio y tapar. Guarde en una caja fuerte de luz, ya sea en 4 ° C o -20 ° C. </li></ol> Parte 6: Imágenes representativas de explantes En un experimento exitoso, el explante y el agregado COS permanecerá junto a la otra y no se alejan como el colágeno conjuntos. Habrá un mínimo de crecimiento excesivo de los axones, el crecimiento excesivo axonal significativo indica que el explante de la columna dorsal se cultivó durante mucho tiempo. No habrá ampollas que crecen en el explante, vesiculación se produce cuando el tejido está en contacto directo con el medio de cultivo. La capacidad de la molécula de señalización para reorientar los axones se evaluó mediante microscopía confocal. La extensión de la reorientación del axón puede ser cuantificado mediante la medición del ángulo medio de la reorientación de los axones más cercano al agregado de células COS 3. Como se muestra en la Figura 1, las células COS que expresan myc-etiquetados BMP7 (azul) cambie la orientación Tag1 + axones de la comisura (verde) de distancia de la fuente de BMP7 (fig. 1B), similar a la manera en que un explante de la placa del techo repele axones de la comisura (Figura 1A). Las células COS que expresan un vector de control no tienen ningún efecto sobre la orientación de los axones comisurales 3,4. Estos explantes se marcaron también con anticuerpos contra el factor de transcripción Isl1 / 2 (rojo), que decora las neuronas motoras y las interneuronas dorsal. Este resultado fue el primer indicio de que un miembro de la familia media en BMP la actividad de la placa del techo chemorepellent 3. <img src="/files/ftp_upload/1853/1853fig1tmb.jpg" border="0" /> Figura 1: Por favor, <a href="https://www.jove.com/files/ftp_upload/1853/1853fig1.jpg">haga clic aquí</a> para ver una versión ampliada de la 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>

Los factores críticos que determinan el éxito en la realización de este ensayo son en primer lugar, el tejido no debe ser entendido con dispasa demasiado prolongada de un período, este tratamiento se traducirá en el tejido a ser muy pegajoso y con disminución de la viabilidad. Dos, el colágeno debe estar perfectamente preparado y mantenerse en hielo tanto como sea posible. Cada vez será más difícil de manejar si se empieza a configurar, es decir, a su vez de color rosa. Tres, la aguja de tungsteno debe estar s...

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

Axones de la comisura dorsal de la médula espinal de vertebrados han sido un modelo de un sistema de valor incalculable en el que para identificar las señales de guía axonal. Aquí se describe un ensayo in vitro &quot;, el ensayo de reorientación&quot;, que se ha utilizado ampliamente para estudiar el efecto de las señales intrínsecas y extrínsecas en la orientación de los axones de la comisura 2. Este ensayo fue desarrollado por numerosas personas en los laboratorios de Jane Dodd, Jessell Thomas y Andrew Lumsden (ver agradecimientos para más detalles) y las versiones de este ensayo se utilizaron para demostrar las actividades de la reorientación de las principales moléculas axón orientación, incluyendo la chemorepellent en el BMP techo de placa de 3,4 y las actividades de chemoattractive Netrin1 5 y Sonic Hedgehog (Shh) 6 en la placa de piso en la médula espinal. Explantes que comprende 2-3 segmentos de la dorsal de dos tercios de la médula espinal se disecan a partir del día embrionario (E) 11 ratas y se cultivaron en tres dimensiones geles de colágeno 7. E11 espinal dorsal explantes contienen recién nacido neuronas comisurales, que pueden ser identificadas por su expresión axonal de la glicoproteína, Tag1 8. En el transcurso de 30-40 horas en la cultura, la trayectoria de los axones comisurales se resume en estas explantes dorsal con un tiempo similar a la observada en vivo. Esta trayectoria axonal puede ser impugnada mediante la colocación de cualquiera de los tejidos de prueba o un agregado de células COS que expresan una molécula de señalización candidato en contacto con uno de los bordes laterales de la explante dorsal. Axones de la comisura que se extienden en los alrededores del tejido adjunta crecerá bajo la influencia de la placa del techo y las señales endógenas de los tejidos laterales ectópico. El grado en que los axones de la comisura se reorientan en estas circunstancias se pueden cuantificar. El uso de este ensayo, es posible tanto a examinar la suficiencia de una señal en particular para reorientar axones de la comisura 3,4 y la necesidad de que esta señal para dirigir la trayectoria de la comisura 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

Este ensayo evalúa la capacidad de una molécula de señalización, la proteína morfogenética ósea 7 aquí (BMP7), para reorientar axones de la comisura. Un explante de la médula espinal dorsal de embriones se cultivaron junto a un conjunto de células secretoras de COS los factores de crecimiento candidato. Reorientado axones de la comisura de crecimiento en el explante se visualizan mediante técnicas de inmunohistoquímica.

Ensayo de la capacidad de difusible moléculas de señalización para reorientar embrionarias axones de la comisura espinal

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

8.8K vistas.  University of Southern California. Este ensayo evalúa la capacidad de una molécula de señalización, la proteína morfogenética ósea 7 aquí (BMP7), para reorientar axones de la comisura. Un explante de la médula espinal dorsal de embriones se cultivaron junto a un conjunto de células secretoras de COS los factores de crecimiento candidato. Reorientado axones de la comisura de crecimiento en el explante se visualizan mediante técnicas de inmunohistoquímica.

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.

Fabricación de un dispositivo de microfluidos para la compartimentación de las neuronas y los axones Soma

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

Investigación

Biología

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.

La médula espinal de Electrofisiología

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.

Cultura de embriones de ratón de pulmón, un sistema para evaluar los mecanismos moleculares de la ramificación

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.

Mediada por adenovirus eliminación de moléculas de señalización genética en cultivos de fibroblastos de embriones de ratón primaria

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 ensayos (FRAP y FLIP) para medir la dinámica de la cromatina de proteínas en células vivas madre embrionarias

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.

Ensayo de la actividad quinasa 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 sistema para la<em&gt; Ex vivo</em&gt; Cultivo de páncreas embrionario

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.

Los explantes blastómeras para la Prueba de Compromiso destino celular durante el desarrollo embrionario

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.

Mecánica estimulación inducida por calcio Propagación de Ondas en monocapas de células: el ejemplo de las especies bovina células endoteliales corneales

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).