Este trabajo es apoyado por becas de investigación de los Institutos Nacionales de Salud (HL068915; HL088206), un Premio al Investigador Nuevo en el Fondo de Investigación Thrasher, y las contribuciones al Fondo de conducción cardiaca en el Hospital de Niños de Boston.

Parte 1: Montar la construcción de moldes de fundición <ol><li> Use una hoja de afeitar para reducir a la mitad el tubo de silicona (VWR) y cortarlo en tres pedazos cm de largo. </li><li> Coloque una gota de RTV adhesivo implantes de silicona de calidad (Rhodia) en el interior de cada extremo de la tubería. </li><li> Rápidamente coloque un pedazo pequeño (1 cm x 1 cm) de malla de poliéster (McMaster-Carr) sobre la caída de adhesivo de silicona y alinearla con el extremo de la tubería. Esto proporcionará una superficie ligeramente elevada y plana para la conexión construir. Repita para el otro extremo. </li><li> Deje que el molde para secar a temperatura ambiente durante 3 días. Es útil para hacer moldes de 20 a 30 a la vez, ya que son esencialmente desechables. </li><li> Una vez que el adhesivo esté completamente seco, almacenar estos moldes en un vaso lleno de etanol al 70% hasta que se necesiten. Este paso ayudará a esterilizar los moldes y el producto terminado se muestra en la Figura 1. </li></ol> Parte 2: Preparación para el aislamiento de células con mioblastos <ol><li> Diluir 1 mg laminina (Sigma) en 250 ml de 0,22 micras, esterilizada por filtración de buffer fosfato salino (PBS) que contiene 1% de penicilina / estreptomicina (Invitrogen) y el 1% Fungizone (Invitrogen). </li><li> Escudo 150 mm placas de cultivo tisular (BD Falcon) con 4 ml de la solución diluida de laminina paso 1.1 e incubar a 37 º C durante al menos 4 horas antes de la galjanoplastia mioblastos esqueléticos primaria. </li><li> Hacer que el medio de mioblastos mediante la mezcla de F-10 de Ham mezcla de nutrientes (Sigma) con 20% de SFB (Atlanta Biologicals), 5 ng / L Factor de crecimiento de fibroblastos (Promega), 1% penicilina / estreptomicina (Invitrogen), y el 1% Fungizone ( Invitrogen). </li><li> Prepare la solución de los músculos esqueléticos digestión mediante la disolución de 1 g de ~ 295 U / mg de colagenasa 2 (Worthington) en 100 ml de 2,4 U / mL Dispasa-2 (Roche). Añadir 0,037 g de CaCl 2 y mezclar bien. Filtro de esterilizar el tampón de digestión con un disco de 0.2 micras de filtro instalado en una jeringa de 10 ml y almacenar alícuotas a -20 ° C. Coloque la cantidad necesaria para la digestión de los tejidos a 37 ° C, junto con los medios de comunicación de mioblastos. </li></ol> Parte 3: Aislamiento de mioblastos de músculo esquelético <ol><li> El uso de pinzas y tijeras pequeñas, eliminar músculos paravertebrales de la muerte neonatal ratas Lewis por corte en la parte posterior de la médula espinal, quitando la piel y las capas de la fascia y los músculos de la escisión. </li><li> En una campana de cultivo de tejidos, el lugar de los músculos extirpado en un tubo cónico de 50 ml (BD Falcon) llena con 40 ml de solución salina Hanks 1X (Balanced HBSS) (Invitrogen) en el hielo. Dejar que las tiras musculares que se depositan en el fondo del tubo y retire con cuidado la mayor parte de los restos de líquido y sangre por succión al vacío con una pipeta Pasteur estéril en una campana de la cultura (Baker). </li><li> Vierta las piezas del músculo recogieron sobre una placa de cultivo de 150 mm y eliminar la mayor cantidad de líquido que queda como sea posible con la succión. Usando dos de un solo filo hojas de afeitar pelos en el tejido hasta formar una pasta espesa. Vierta precalentado solución de digestión de la pasta y la transferencia de la mezcla a un tubo de 50 ml fresca con una pipeta de 10 ml. Durante la digestión enzimática, el tejido es de vez en cuando triturado (sin burbujas) con una pipeta de 10 mL equipado en un teléfono inalámbrico con una pipeta-Aid (Drummond). Una vez más, permitir que el tejido para resolver y eliminar el exceso de líquido. </li><li> Coloque el tubo en una plataforma oscilante y digerir a 37 º C durante unos 30 minutos. </li><li> Una vez que el músculo está licuado, pasar a través de un colador celular 70 micras (BD Falcon) y se centrifuga a temperatura ambiente durante 10 minutos a 600 xg (Beckman GP). Con una pipeta de Pasteur y de succión, retire la mayor cantidad de líquido posible y resuspender el precipitado con 10 ml de medio de mioblastos caliente. </li><li> Pre-placa de las células en una placa de 150 mm sin recubrimiento de cultivo de tejidos durante 15 minutos para ayudar a eliminar los fibroblastos. </li><li> Transferencia de las células que aún no han conectado a la cultureware laminina recubierto y se diluye de modo que no es un plato para cada 1 a 2 crías de rata. Colocar la placa en una incubadora humidificada conjunto a 37 º C con CO2 al 5%. </li></ol> Parte 4: Usar las construcciones de ingeniería tisular (5 ml) <ol><li> Después de 1 a 2 días, retire 10 de los 150 mm placas de cultivo de tejidos de mioblastos de la incubadora, lavar dos veces con 37 ° C PBS 1X, y añadir 4 ml de agua tibia 0,05% de tripsina-EDTA a cada plato. Retorno de las placas a la incubadora durante 5 min. </li><li> Cosecha de los mioblastos separado de las placas en la cubierta la cultura y recoger el líquido en un tubo de 50 ml (BD Falcon). Utilizar 10 ml de medio de mioblastos para lavar todos los platos y combinar esto con las células en el tubo de centrífuga. Que sedimenten las células a temperatura ambiente durante 10 minutos a 600 xg (Beckman GP) y eliminar el exceso de líquido como se describe en el paso 3.5. </li><li> Resuspender las células con mioblastos en 1,6 ml de medio de mioblastos y el lugar en el hielo. </li><li> Con unas pinzas, retire con cuidado la construcción de 4 moldes de paso 1,5 y enjuague dos veces con PBS 1X. Vacío de aspiración de todos los rastros de PBS con una pipeta Pasteur antes de colocar cada molde en un cu tejidos de 6 pocilloslture placa (BD Falcon). </li><li> Reúna a los otros reactivos necesarios para la preparación de la construcción (F10 Jamón de 10X, los antibióticos, el colágeno tipo 1 [3,9 mg / ml] [6] y Matrigel ™) en el hielo y mantener los medios de comunicación de mioblastos a 37 º C. Combine 500 l 10 veces más de Ham F10, 100 l de penicilina / estreptomicina, 100 Fungizone l, 1,6 mL de colágeno, y 750 ™ l Matrigel en un tubo cónico de 15 ml. Mezclar la solución durante 10 segundos utilizando una Mini-vórtice (VWR) establece en 10 y el lugar en el hielo. </li><li> A continuación, añadir 80 l de NaHCO3 y la suspensión de mioblastos a partir del paso de 3,3 a la mezcla del paso 3.5. Vortex toda la mezcla durante 10 segundos y volver el tubo al cubo de hielo durante unos minutos para limpiar la solución de burbujas. </li><li> Con cuidado, tire la mezcla en los moldes antes de que comience a solidificarse con una pipeta de 10 ml. Cada molde tiene capacidad para aproximadamente 1 ml de líquido viscoso. Trate de evitar la introducción de burbujas de aire durante este procedimiento. </li><li> Transferir cuidadosamente la placa que contiene las construcciones de fundición a la incubadora. Después de aproximadamente 30 minutos, retire las construcciones solidificado de la incubadora y añadir con cuidado a cada medio de mioblastos y para cubrir el tejido construir. Devolver la placa a la incubadora (Figura 2). </li></ol> Parte 5: Los resultados representativos Cuando este protocolo se ejecuta correctamente, la construcción del tejido de mioblastos que contienen está listo para la implantación in vivo (es decir, cuando se saca del molde) o más en los análisis in vitro después de 2 días. <img src="/files/ftp_upload/1137/Figure1.jpg" alt="" /> Figura 1. Ejemplos de completar la construcción de moldes (ver Paso 1.5). <img src="/files/ftp_upload/1137/Figure2.jpg" alt="" width="400" height="321" /> Figura 2. A solidificado mioblastos tejido que contiene la construcción [1]. <img src="/files/ftp_upload/1137/Figure3.jpg" alt="" /> Figura 3. Perfiles en H &amp; E manchadas de un tejido que contiene la construcción de mioblastos. &quot;A&quot; representa un corte longitudinal y &quot;B&quot; muestra una sección transversal.

<ol>
	<li>Choi, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+conduction+through+engineered+tissue.">Cardiac conduction through engineered tissue.</a> Am J Pathol. 169 (1), 72-85 (2006).</li><li>Rando, T. A., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+mouse+myoblast+purification,+characterization,+and+transplantation+for+cell-mediated+gene+therapy.">Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy.</a> J Cell Biol. 125 (6), 1275-1287 (1994).</li><li>Blau, H. M., Webster, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+human+muscle+cells.">Isolation and characterization of human muscle cells.</a> Proc Natl Acad Sci U S A. 78 (9), 5623-5627 (1981).</li><li>Powell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+human+bioartificial+muscles+expressing+a+foreign+recombinant+protein+for+gene+therapy.">Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy.</a> Hum Gene Ther. 10 (4), 565-577 (1999).</li><li>Vandenburgh, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+skeletal+muscle+organoids+for+reversible+gene+therapy.">Tissue-engineered skeletal muscle organoids for reversible gene therapy.</a> Hum Gene Ther. 7 (17), 2195-2200 (1996).</li><li>Gallop, P. M., Seifter, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+Properties+of+Soluble+Collagens.">Preparation and Properties of Soluble Collagens.</a> Methods in Enzymology. 6, 635-641 (1963).</li></ol>

Los experimentos con animales se realizaron de acuerdo con las directrices y normas establecidas por el Cuidado de Animales institucional y el empleo en el Hospital de Niños de Boston.

Los moldes en los que será la construcción de tejido reparto se puede hacer en cualquier forma y tamaño, sin embargo, es necesario que haya al menos dos puntos de fijación. De lo contrario, la matriz y las células forman una estructura esférica y las células mueren. En el presente Protocolo, se describe el uso de una malla de poliéster para este propósito, sin embargo, también hemos utilizado con éxito una malla de acero inoxidable. Obviamente, los moldes más grandes requieren más células, y un mayor volum...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Silicone tubing</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>60985-724</td>
<td>&nbsp;</td>
<tr>
<td>Silicone adhesive</td>
<td>&nbsp;</td>
<td>Rhodia Silicones</td>
<td>MED ADH 4300 RTV</td>
<td>&nbsp;</td>
<tr>
<td>Polyester Mesh</td>
<td>&nbsp;</td>
<td>McMaster-Carr</td>
<td>93185T17</td>
<td>&nbsp;</td>
<tr>
<td>Laminin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L2020</td>
<td>&nbsp;</td>
<tr>
<td>Nutrient Mixture F-10 HAM</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>N6908</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Atlanta Biologicals</td>
<td>S11550</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Fungizone</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15290-018</td>
<td>&nbsp;</td>
<tr>
<td>Dispase-2</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>10295825001</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase 2</td>
<td>&nbsp;</td>
<td>Worthington</td>
<td>46H8863</td>
<td>&nbsp;</td>
<tr>
<td>Basic Fibroblast Growth Factor</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>G5071</td>
<td>&nbsp;</td>
<tr>
<td>150 mm tissue culture dishes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>353025</td>
<td>&nbsp;</td>
<tr>
<td>0.05% (1X) Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25300</td>
<td>&nbsp;</td>
<tr>
<td>1X Hanks Balanced Salt Solution</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14170-112</td>
<td>&nbsp;</td>
<tr>
<td>7.5% NaHCO3</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25080-094</td>
<td>&nbsp;</td>
<tr>
<td>70 &mu;m cell strainer</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>6-well plates</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>353046</td>
<td>&nbsp;</td>
<tr>
<td>50 mL Conical Vial</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352098</td>
<td>&nbsp;</td>
<tr>
<td>15 mL Conical Vial</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352099</td>
<td>&nbsp;</td>
<tr>
<td>0.2 &mu;m filter</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>194-2520</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

fabrication of myogenic engineered tissue constructs

A pesar de que los marcapasos electrónicos para salvar vidas los dispositivos médicos, su rendimiento a largo plazo en pacientes pediátricos puede ser problemática debido a las restricciones impuestas por el tamaño pequeño de un niño y su crecimiento inevitable. En consecuencia, hay una verdadera necesidad de terapias innovadoras diseñadas específicamente para los pacientes pediátricos con trastornos del ritmo cardíaco. Proponemos que una alternativa conductor biológica que consiste en una matriz a base de colágeno que contiene autologously células derivadas de una mejor podrían adaptarse al crecimiento, reducir la necesidad de cirugías recurrente, y en gran medida a mejorar la calidad de vida de estos pacientes. En el presente estudio, se describe un procedimiento para la incorporación de cultivos primarios de células con mioblastos esqueléticos dentro de una matriz de hidrogel para diseñar una construcción de tejido quirúrgicamente implantable que servirá como un conducto eléctrico entre las cámaras superior e inferior del corazón. En última instancia, esperamos con este tipo de ingeniería tisular para restablecer la conducción eléctrica auriculoventricular en los niños con bloqueo cardíaco completo. En vista de ello, aislamos los mioblastos de músculo esquelético de ratas Lewis neonatal y la placa que en laminina recubierto platos de cultivo de tejidos utilizando una versión modificada de los protocolos establecidos<sup&gt; [2, 3]</sup&gt;. Después de uno o dos días, las células cultivadas se recogen y se mezclan con los antibióticos, el colágeno tipo 1, Matrigel ™, y NaHCO<sub&gt; 3</sub&gt;. El resultado es una solución viscosa, uniforme que se puede convertir en un molde de casi cualquier forma y tamaño<sup&gt; [1, 4, 5]</sup&gt;. Para construcciones de nuestro tejido, empleamos el colágeno tipo 1 aisladas de piel de cordero fetal mediante el procedimiento habitual<sup&gt; [6]</sup&gt;. Una vez que el tejido ha consolidado a 37 ° C, los medios de cultivo se agrega cuidadosamente a la plancha hasta que la construcción se encuentra sumergido. La ingeniería tisular se deja condensar aún más a través de la deshidratación durante 2 días más, momento en el que está listo para<em&gt; In vitro</em&gt; Evaluación o implantación quirúrgica.

Watch this Scientific Journal Video about Fabrication of Myogenic Engineered Tissue Constructs at JoVE.com

Fabrication of Myogenic Engineered Tissue Constructs

Este sentido, demuestran la fabricación de estructuras a base de colágeno del tejido, con mioblastos esqueléticos. Estas construcciones de ingeniería 3-D se puede utilizar para sustituir o reparar los tejidos<em&gt; In vivo</em&gt;. Para nuestros propósitos, hemos diseñado estos como un canal atrioventricular eléctricos para la reparación de un bloqueo cardíaco completo<sup&gt; [1]</sup&gt;.

Fabricación de construcciones de ingeniería tisular miogénica

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

fabrication-of-myogenic-engineered-tissue-constructs

Research

JoVE Journal

Biology

10.4K vistas.  Children's Hospital Boston and Harvard Medical School. Este sentido, demuestran la fabricación de estructuras a base de colágeno del tejido, con mioblastos esqueléticos. Estas construcciones de ingeniería 3-D se puede utilizar para sustituir o reparar los tejidos In vivo. Para nuestros propósitos, hemos diseñado estos como un canal atrioventricular eléctricos para la reparación de un bloqueo cardíaco completo [1].

Video: Fabrication of Myogenic Engineered Tissue Constructs

PDMS Device Fabrication and Surface Modification

PDMS fabricación de dispositivos y modificación de la superficie

Investigación

Biología

Fabrication of the Thermoplastic Microfluidic Channels

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using polymer/nanoparticle composite microscale lysis and solid phase extraction columns. The recovered samples are concentrated, small volume samples that are PCRable, without any additional cleanup. Here, we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then, we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns. We demonstrate grafting and polymerization of a carbon nanotube/polymer composite column for bacterial cell lysis. We then show the lysis process followed by solid phase extraction of nucleic acids from the sample on chip using a silica/polymer composite column. The attached protocols contain detailed instructions on how to make both lysis and solid phase extraction columns.

Here we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns.

Fabricación de los canales de microfluidos termoplástico

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using ...

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Actina Co-sedimentación de ensayo, pues el análisis de proteína de unión a la F-actina

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Fabrication of Amperometric Electrodes

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we describe the techniques needed to generate low noise (&lt;0.5 pA) electrodes.  The techniques have been modified from published descriptions by previous researchers (1,2).  Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber.  Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip.  The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal.  Lastly, the electrodes are placed in an oven to cure the epoxy.  Careful handling of the electrodes is critical to ensure that they are made consistently and without damage.  This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells.

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

Fabricación de electrodos amperométricos

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we ...

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Implantación de la ingeniería tisular en el corazón de rata

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Un sistema para el cultivo de células epiteliales del pigmento del iris para el Estudio de Regeneración del objetivo de Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

Comparación cuantitativa de Cis-Elemento regulador (CRE) Las actividades de transgénicos Drosophila melanogaster</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

Criopreservación de espermatozoides de ratón y de recuperación con el I · Kit de Cryo

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, distinct microenvironments can impose differentiation, migration, and proliferation phenotypes, and on the tissue level the microenvironment processes as complex as morphogenesis and tumorigenesis1. Not only do the cell and molecular contents of microenvironments impact the cells within, but so do the elasticity2 and geometry3 of the tissue. Defined as the sum total of cell-cell, -ECM, and -soluble factor interactions, in addition to physical characteristics, the microenvironment is complex. The phenotypes of cells within a tissue are partially due to their genomic content and partially due to the combinatorial interactions with the microenviroment. A major challenge is to link specific combinations of microenvironmental components with distinctive behaviours.
Here, we present the microenvironment microarray (MEArray) platform for cell-based functional screening of interactions with combinatorial microenvironments4. The method allows for simultaneous control of the molecular composition and the elastic modulus, and combines the use of widely available microarray and micropatterning technologies. MEArray screens require as few as 10,000 cells per array, which facilitates functional studies of rare cell types such as adult progenitor cells. A limitation of the technology is that entire tissue microenvironments cannot be completely recapitulated on MEArrays. However, comparison of responses in the same cell type to numerous related microenvironments, for instance pairwise combinations of ECM proteins that characterize a given tissue, will provide insights into how microenvironmental components elicit tissue-specific functional phenotypes.
MEArrays can be printed using a wide variety of recombinant growth factors, cytokines, and purified ECM proteins, and combinations thereof. The platform is limited only by the availability of specific reagents. MEArrays are amenable to time-lapsed analysis, but most often are used for end point analyses of cellular functions that are measureable with fluorescent probes. For instance, DNA synthesis, apoptosis, acquisition of differentiated states, or production of specific gene products are commonly measured. Briefly, the basic flow of an MEArray experiment is to prepare slides coated with printing substrata and to prepare the master plate of proteins that are to be printed. Then the arrays are printed with a microarray robot, cells are allowed to attach, grow in culture, and then are chemically fixed upon reaching the experimental endpoint. Fluorescent or colorimetric assays, imaged with traditional microscopes or microarray scanners, are used to reveal relevant molecular and cellular phenotypes (Figure 1).

Fabrication and Use of MicroEnvironment microArrays MEArrays

A combinatorial functional screening method for gaining insights into the impacts of the molecular composition of microenvironments on cellular functions is described. The method takes advantage of existing microarray-based technologies to generate arrays of defined combinatorial microenvironments that support cell adhesion and functional analysis.

Fabricación y uso de microarrays microambiente (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

Recombinería homólogos Construcciones recombinación en<em&gt; Drosophila</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...