Ce travail est soutenu par des subventions de recherche du National Institutes of Health (HL068915; HL088206), un Prix du nouveau chercheur du Fonds de recherche Thrasher, et les contributions au Fonds de la conduction cardiaque au Centre hospitalier pour enfants de Boston.

Partie 1: Réunir construire des moules de fonderie <ol><li> Utilisez une lame de rasoir pour réduire de moitié tube en silicone (VWR) et le couper en 3 morceaux cm de long. </li><li> Déposer une goutte d&#39;implant de qualité adhésive silicone RTV (Rhodia) à l&#39;intérieur de chaque extrémité du tube. </li><li> Vite placer un petit morceau (1 cm x 1 cm) de filet de polyester (McMaster-Carr) sur la chute d&#39;adhésif de silicone et de l&#39;aligner avec le bout du tuyau. Cela fournira une surface légèrement surélevé et plate pour la fixation de construire. Répétez l&#39;opération pour l&#39;autre extrémité. </li><li> Laisser le moule à sécher à température ambiante pendant 3 jours. Il est utile de faire 20 à 30 moules à la fois, que ce sont essentiellement à usage unique. </li><li> Une fois la colle est complètement sèche, de stocker ces moules dans un bécher rempli avec de l&#39;éthanol à 70% jusqu&#39;à ce qu&#39;ils soient nécessaires. Cette étape permettra de stériliser les moules et le produit fini est montré dans la figure 1. </li></ol> Partie 2: Préparation pour l&#39;isolation des myoblastes <ol><li> Diluer 1 mg laminine (Sigma) dans 250 ml de 0,22 um, stérilisée par filtration tampon phosphate salin (PBS) contenant 1% de pénicilline / streptomycine (Invitrogen) et Fungizone 1% (Invitrogen). </li><li> Manteau plaques de 150 mm de culture de tissu (BD Falcon) avec 4 ml de la solution diluée laminine partir de l&#39;étape 1.1 et incuber à 37 º C pendant au moins 4 heures avant l&#39;étalement des myoblastes squelettiques primaires. </li><li> Faire le support myoblastes en mélangeant Ham F-10 mélange nutritif (Sigma) avec du FBS à 20% (Atlanta Biologicals), 5 ng / L facteur de croissance basique des fibroblastes (Promega), 1% de pénicilline / streptomycine (Invitrogen), et Fungizone 1% ( Invitrogen). </li><li> Préparer la solution du muscle squelettique de digestion en dissolvant 1 g de ~ 295 U / mg collagénase 2 (Worthington) dans 100 ml 2,4 U / ml Dispase-2 (Roche). Ajouter 0,037 g de CaCl 2 et bien mélanger. Filtre stériliser le tampon de digestion en utilisant un disque filtre de 0,2 um monté sur une seringue de 10 ml et des aliquots conserver à -20 ° C. Placez la quantité nécessaire pour la digestion des tissus à 37 ° C avec les médias des myoblastes. </li></ol> Partie 3: isolement des myoblastes du muscle squelettique <ol><li> En utilisant des pinces et des petits ciseaux, retirez muscles spinaux de rats Lewis morts néonatales par tranchage en bas du dos le long de la moelle épinière, éplucher la peau et les couches du fascia puis exciser les muscles. </li><li> Dans une hotte de culture de tissus, placer les muscles excisés dans un tube conique de 50 ml (BD Falcon) rempli avec 40 ml de 1X Hanks Balanced Salt Solution (HBSS) (Invitrogen) sur la glace. Laisser les bandelettes de muscle pour se déposent au fond du tube et retirez soigneusement la plupart des restes de liquide et de sang par aspiration sous vide avec une pipette stérile Pasteur dans une hotte de la culture (Baker). </li><li> Verser les morceaux de muscle récoltés sur une boîte de culture de 150 mm et enlever le plus de liquide restant possible avec aspiration. En utilisant deux lames de rasoir seul tranchant émincer les tissus d&#39;une pâte épaisse. Verser la solution de digestion préchauffé sur la pâte et transférer le mélange dans un nouveau tube de 50 ml à l&#39;aide d&#39;une pipette de 10 ml. Pendant la digestion enzymatique, le tissu est parfois triturés (sans bulles) avec une pipette 10 ml monté sur un fil pipette-Aid (Drummond). Encore une fois, laisser le tissu de régler et retirer l&#39;excès de liquide. </li><li> Placer le tube sur une plateforme à bascule et digérer à 37 º C pendant environ 30 min. </li><li> Une fois que le muscle est liquéfié, passer la solution à travers une passoire cellule 70 um (BD Falcon) et centrifuger à température ambiante pendant 10 min à 600 xg (Beckman GP). Avec une pipette Pasteur et d&#39;aspiration, enlever autant de liquide que possible et remettre le culot avec 10 mL de milieu de myoblastes chaud. </li><li> Pré-plaque de cellules sur une plaque de culture non couché 150 mm de tissu pendant 15 minutes pour aider à éliminer les fibroblastes. </li><li> Transférer les cellules qui n&#39;ont pas encore attaché à la laminine cultureware enduits et diluer afin qu&#39;il y est une plaque pour tous les 1 à 2 chiots rat. Placer la plaque dans un ensemble incubateur humidifié à 37 ° C avec 5% de CO 2. </li></ol> Partie 4: Moulage des constructions de l&#39;ingénierie tissulaire (5 ml) <ol><li> Après 1 à 2 jours, retirer 10 des 150 plaques myoblastes de culture tissulaire mm de l&#39;incubateur, les rincer deux fois avec 37 ° C PBS 1X, et ajouter 4 ml d&#39;eau tiède 0,05% de trypsine-EDTA à chaque plaque. Retour des plaques dans l&#39;incubateur pendant 5 min. </li><li> Récolter les myoblastes détachées des plaques dans la hotte de la culture et de recueillir le liquide dans un tube de 50 ml (BD Falcon). Utiliser 10 mL de milieu de myoblastes pour le rinçage de toutes les plaques et de combiner cela avec les cellules dans le tube à centrifuger. Pellet les cellules à température ambiante pendant 10 min à 600 xg (Beckman GP) et enlever l&#39;excès de liquide tel que décrit dans l&#39;étape 3.5. </li><li> Resuspendre les cellules myoblastes dans 1,6 mL de milieu de myoblastes et les placer sur la glace. </li><li> En utilisant une pince, retirez soigneusement 4 moules de construire de l&#39;étape 1.5 et les rincer deux fois avec du PBS 1X. Aspiration hors toute trace de PBS avec une pipette Pasteur avant de placer chaque moule dans une cu tissus à 6 puitsPlaque LTURE (BD Falcon). </li><li> Rassemblez les autres réactifs nécessaires pour la préparation de la construction (F10 de Ham 10X, les antibiotiques, collagène de type 1 [3,9 mg / ml] [6] et de Matrigel ™) sur la glace et de garder les médias myoblastes à 37 º C. Combinez 500 ul 10X Ham F10, 100 uL de pénicilline / streptomycine, 100 Fungizone ul, 1,6 ml de collagène, et 750 ™ Matrigel uL dans un tube 15 ml conique. Mélanger la solution pendant 10 secondes en utilisant un mini-Vortexer (VWR) fixé à 10 et les placer sur la glace. </li><li> Ensuite, ajouter 80 ul de NaHCO 3 et la suspension de l&#39;étape myoblastes 3,3 à mélange de l&#39;étape 3.5. Vortex l&#39;ensemble du mélange pendant 10 secondes et retourner le tube pour le seau à glace pendant quelques minutes pour effacer la solution de bulles. </li><li> Soigneusement coulée le mélange dans les moules avant qu&#39;il commence à se solidifier à l&#39;aide d&#39;une pipette de 10 ml. Chaque moule peut contenir environ 1 mL du liquide visqueux. Essayez d&#39;éviter d&#39;introduire des bulles d&#39;air lors de cette procédure. </li><li> Soigneusement transfert de la plaque contenant les constructions exprimées à l&#39;incubateur. Après ~ 30 min, retirez les constructions solidifié de l&#39;incubateur et ajouter prudemment moyennes myoblastes dans chaque puits afin de couvrir le tissu construire. Retour à la plaque de l&#39;incubateur (figure 2). </li></ol> Partie 5: Les résultats représentatifs Lorsque ce protocole est correctement exécutée, la construction des tissus contenant des myoblastes est prêt pour implantation in vivo (c&#39;est à dire lorsqu&#39;il est retiré du moule) ou pour d&#39;autres analyses in vitro après 2 jours. <img src="/files/ftp_upload/1137/Figure1.jpg" alt="" /> Exemples Figure 1. Des moules achevée de construire (voir l&#39;étape 1.5). <img src="/files/ftp_upload/1137/Figure2.jpg" alt="" width="400" height="321" /> Figure 2. Un solidifié myoblastes tissu contenant de construire [1]. <img src="/files/ftp_upload/1137/Figure3.jpg" alt="" /> Figure 3. Sections H &amp; E teinté d&#39;un tissu contenant des myoblastes construire. «A» représente une coupe longitudinale et &quot;B&quot; montre une section transversale.

<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>

Expériences sur les animaux ont été effectuées en conformité avec les directives et les règlements énoncés par le soin des animaux et du Comité institutionnel utilisation à l&#39;Hôpital pour enfants de Boston.

Les moules dans lesquels la construction des tissus seront jetés peuvent être faites dans n&#39;importe quelle forme et taille, mais il doit y avoir au moins deux points de fixation. Sinon, la matrice et les cellules forment une structure sphérique et les cellules meurent. Dans le présent protocole, nous décrivons l&#39;utilisation d&#39;un maillage de polyester à cet effet, mais nous avons aussi utilisé avec succès maille d&#39;acier inoxydable. Évidemment, les grands moules, il faudra plus de cellules et un p...

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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

Malgré le fait que les stimulateurs électroniques sont des dispositifs de sauvetage médical, leur performance à long terme chez les patients pédiatriques peut être problématique du fait des restrictions imposées par la petite taille de l&#39;enfant et leur croissance inévitable. Par conséquent, il ya un véritable besoin pour des thérapies innovantes conçues spécifiquement pour les patients pédiatriques atteints de troubles du rythme cardiaque. Nous proposons qu&#39;une alternative conducteur biologiques constitués d&#39;une matrice à base de collagène autologue contenant des cellules dérivées pourrait mieux s&#39;adapter à la croissance, réduire le besoin de chirurgies récurrentes, et d&#39;améliorer grandement la qualité de vie de ces patients. Dans la présente étude, nous décrivons une procédure pour intégrer primaires du squelette des cultures de cellules myoblastes dans une matrice d&#39;hydrogel à la mode d&#39;une construction des tissus implantables chirurgicalement qui servira comme un conduit électrique entre les chambres supérieures et inférieures du cœur. Finalement, nous prévoyons d&#39;utiliser ce type d&#39;ingénierie tissulaire pour restaurer la conduction électrique auriculo-ventriculaire chez les enfants présentant un bloc auriculo-ventriculaire complet. Compte tenu de cela, nous isolons les myoblastes dans les muscles squelettiques de rats Lewis néonatale et la plaque-les sur la laminine revêtement des boîtes de culture tissulaire en utilisant une version modifiée de protocoles établis<sup&gt; [2, 3]</sup&gt;. Après un à deux jours, les cellules cultivées sont collectés et mélangés avec les antibiotiques, collagène de type 1, Matrigel ™, et NaHCO<sub&gt; 3</sub&gt;. Le résultat est une solution visqueuse uniforme qui peut être coulé dans un moule de près toute forme et taille<sup&gt; [1, 4, 5]</sup&gt;. Pour nos constructions des tissus, nous employons de collagène de type 1 isolées de la peau d&#39;agneau du foetus en utilisant des procédures standard<sup&gt; [6]</sup&gt;. Une fois le tissu a solidifié à 37 ° C, la culture médiatique est soigneusement ajouté à la plaque jusqu&#39;à ce que la construction est submergé. L&#39;ingénierie tissulaire est alors autorisé à se condenser davantage grâce à la déshydratation pour les 2 jours de plus, à quel point il est prêt pour<em&gt; In vitro</em&gt; L&#39;évaluation ou de chirurgie d&#39;implantation.

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

Fabrication of Myogenic Engineered Tissue Constructs

Ici, nous démontrons la fabrication de base de collagène, le tissu des constructions contenant myoblastes squelettiques. Ces constructions en 3-D modifiés peuvent être utilisés pour remplacer ou réparer des tissus<em&gt; In vivo</em&gt;. Pour nos fins, nous avons conçu ces comme un conduit auriculo-ventriculaire électriques pour la réparation de bloc cardiaque complet<sup&gt; [1]</sup&gt;.

Fabrication de constructions myogéniques de l&#39;ingénierie tissulaire

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 vues.  Children's Hospital Boston and Harvard Medical School. Ici, nous démontrons la fabrication de base de collagène, le tissu des constructions contenant myoblastes squelettiques. Ces constructions en 3-D modifiés peuvent être utilisés pour remplacer ou réparer des tissus In vivo. Pour nos fins, nous avons conçu ces comme un conduit auriculo-ventriculaire électriques pour la réparation de bloc cardiaque complet [1].

Vidéo: Fabrication of Myogenic Engineered Tissue Constructs

PDMS Device Fabrication and Surface Modification

Fabrication de dispositifs en PDMS et modification de surface

Recherche

Biologie

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.

La fabrication des canaux microfluidiques thermoplastique

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.

Co-actine sédimentation Assay; pour l&#39;analyse des Binding Protein à F-actine

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.

Fabrication d&#39;électrodes ampérométriques

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.

Implantation de l&#39;ingénierie tissulaire dans le coeur de rat

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

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.

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

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.

Cryoconservation du sperme de souris et de récupération utilisant les I · 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 ...

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.

Recombineering homologues constructions de recombinaison<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 ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

Préparation du primaire Myogenic précurseurs cellulaires / cultures de myoblastes de basales vertébrés lignées

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.

Phage médiée Livraison de Targeted ARNs Construit pour faire tomber l&#39;expression génique dans<em&gt; E. coli</em

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

Dissection de l&#39;Auditory Bulla chez les souris postnatale: Isolation des os de l&#39;oreille moyenne et l&#39;analyse histologique

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...