Questo lavoro è sostenuto da borse di ricerca dal National Institutes of Health (HL068915; HL088206), un premio Ricercatore Nuovo dal Fondo di ricerca Thrasher, e contributi al fondo di conduzione cardiaca presso il Children Hospital di Boston.

Parte 1: Assemblare costruire stampi <ol><li> Utilizzare una lama di rasoio di dimezzare tubi in silicone (VWR) e tagliarla in 3 pezzi cm di lunghezza. </li><li> Mettere una goccia di protesi in silicone RTV di qualità adesiva (Rhodia) all&#39;interno di ciascuna estremità del tubo. </li><li> Rapidamente posto un piccolo pezzo (1 cm x 1 cm) di poliestere mesh (McMaster-Carr) sulla caduta di silicone e allinearlo con l&#39;estremità del tubo. Questo presenta una superficie leggermente rialzato e piano per il fissaggio costruire. Ripetere l&#39;operazione per l&#39;altra estremità. </li><li> Lasciare lo stampo ad asciugare a temperatura ambiente per 3 giorni. E &#39;utile fare 20-30 stampi in un momento, in quanto questi sono essenzialmente usa e getta. </li><li> Una volta che l&#39;adesivo è completamente asciutto, conservare questi stampi in un bicchiere pieno di etanolo al 70% fino a quando non sono necessari. Questa operazione aiuterà sterilizzare gli stampi e il prodotto finito è mostrata in Figura 1. </li></ol> Parte 2: Preparazione per l&#39;isolamento delle cellule mioblasti <ol><li> Diluire 1 mg Laminina (Sigma) in 250 ml 0,22 micron, sterilizzata per filtrazione tampone fosfato salino (PBS) contenente 1% di penicillina / streptomicina (Invitrogen) e 1% Fungizone (Invitrogen). </li><li> Cappotto 150 millimetri di tessuto piastre di coltura (BD Falcon) con 4 mL di soluzione diluita Laminina dal punto 1.1 e incubare a 37 ° C per almeno 4 ore prima della placcatura primaria mioblasti scheletrici. </li><li> Fai il medium mioblasti mescolando F-10 Ham Miscela di nutrienti (Sigma) con 20% FBS (Atlanta Biologicals), 5 ng / L di base del fattore di crescita dei fibroblasti (Promega), 1% penicillina / streptomicina (Invitrogen), e 1% Fungizone ( Invitrogen). </li><li> Preparare la soluzione di digestione del muscolo scheletrico sciogliendo 1 g di ~ 295 U / mg Collagenasi 2 (Worthington) in 100 ml 2,4 U / mL dispasi-2 (Roche). Aggiungere 0,037 g di CaCl 2 e mescolare bene. Filtro sterilizzare il buffer digestione utilizzando un disco di 0,2 micron filtro montato su una siringa da 10 ml e aliquote conservare a -20 ° C. Posizionare l&#39;importo necessario per la digestione dei tessuti a 37 ° C insieme con i media mioblasti. </li></ol> Parte 3: isolamento dei mioblasti di muscolo scheletrico <ol><li> Con pinze e forbici piccole, rimuovere i muscoli paravertebrali da ratti neonati morti Lewis dal taglio lungo la schiena lungo il midollo spinale, togliendo la pelle e gli strati fascia e poi asportare i muscoli. </li><li> In una cappa colture di tessuti, i muscoli asportato posto in un tubo da 50 ml coniche (BD Falcon) riempito con 40 mL di soluzione 1X Salt Hanks bilanciata (HBSS) (Invitrogen) su ghiaccio. Lasciare le strisce muscolari di depositarsi sul fondo della provetta e rimuovere con attenzione la maggior parte dei residui liquidi e sangue da vuoto di aspirazione con una pipetta Pasteur sterile in una cappa cultura (Baker). </li><li> Versare i pezzi di muscolo raccolte su di un piatto di cultura 150 mm e rimuovere la maggior quantità di liquido residuo possibile con aspirazione. Utilizzando 2 singolo taglio lame di rasoio tritare il tessuto di una pasta spessa. Versare preriscaldata soluzione digestione sulla pasta e trasferire il composto per un nuovo tubo da 50 ml con una pipetta 10 ml. Durante la digestione enzimatica, il tessuto è occasionalmente triturato (senza bolle) con una pipetta 10 mL montata su un cordless Pipetta-Aid (Drummond). Ancora una volta, permette al tessuto di stabilirsi e di rimuovere il liquido in eccesso. </li><li> Posizionare il tubo su una piattaforma a dondolo e digerire a 37 º C per circa 30 min. </li><li> Una volta che il muscolo è liquefatto, filtrare la soluzione attraverso un colino cellula 70 micron (BD Falcon) e centrifugare a temperatura ambiente per 10 min a 600 xg (Beckman GP). Con una pipetta Pasteur e di aspirazione, rimuovere la maggior quantità di liquido possibile e risospendere il precipitato con 10 ml di medium mioblasti caldo. </li><li> Pre-piastra le cellule su una piastra di coltura non patinata 150 millimetri di tessuto per 15 minuti per contribuire a rimuovere fibroblasti. </li><li> Trasferire le cellule che non hanno ancora attaccato al laminina rivestite cultureware e diluire in modo che vi è una piastra per ogni 1 o 2 cuccioli di ratto. Porre la piastra in una serie incubatore umidificato a 37 ° C con 5% di CO 2. </li></ol> Parte 4: Casting costruzioni di ingegneria tessutale (5 ml) <ol><li> Dopo 1 o 2 giorni, rimuovere 10 dei 150 lastre tessuto millimetri cultura mioblasti dal termostato, lavare due volte con 37 ° C 1X PBS e aggiungere 4 ml di caldo 0,05% tripsina-EDTA per ogni piatto. Ritorna le piastre per l&#39;incubatore per 5 min. </li><li> Raccolto il mioblasti staccato dalle piastre nel cofano della cultura e raccogliere il liquido in un tubo da 50 ml (BD Falcon). Usare 10 ml di medium mioblasti a lavare tutti i piatti e combinare questo con le cellule in provetta da centrifuga. Agglomerare le cellule a temperatura ambiente per 10 min a 600 xg (Beckman GP) e rimuovere il liquido in eccesso come descritto al punto 3.5. </li><li> Risospendere le cellule mioblasti in 1,6 ml di terreno mioblasti e posto sul ghiaccio. </li><li> Utilizzando cautela il numero 4 stampini da costruire passo 1,5 e lavare due volte con PBS 1X. Aspirazione aspirare tutte le tracce di PBS con una pipetta Pasteur prima di effettuare ogni stampo in un 6-ben cu tessutolture piastra (BD Falcon). </li><li> Raccogliere le altri reagenti necessari per la preparazione di costruire (F10 Ham di 10X, gli antibiotici, il collagene di tipo 1 [3,9 mg / ml] [6] e Matrigel ™) su ghiaccio e tenere i media mioblasti a 37 ° C. Combina 500 microlitri 10X Ham F10, 100 ul penicillina / streptomicina, 100 microlitri Fungizone, 1,6 ml di collagene, e 750 ™ Matrigel microlitri in un tubo da 15 ml. Miscelare la soluzione per 10 secondi con un Mini-vortex (VWR) impostato su 10 e posto sul ghiaccio. </li><li> Successivamente, aggiungere 80 ml ​​di NaHCO 3 e la sospensione mioblasti dal punto 3.3 a miscela dal punto 3.5. Vortice della miscela per 10 secondi e riportare il tubo al secchio del ghiaccio per un paio di minuti per eliminare la soluzione di bolle. </li><li> Accuratamente fusa il composto negli stampi prima che inizi a solidificare con una pipetta 10 ml. Ogni stampo terrà circa 1 ml di liquido viscoso. Cercate di evitare l&#39;introduzione di bolle d&#39;aria durante questa procedura. </li><li> Trasferire accuratamente l&#39;piastra contenente i costrutti cast dell&#39;incubatore. Dopo circa 30 minuti, togliere i costrutti solidificato dal termostato ed aggiungere con cautela medio mioblasti in ciascun pozzetto per coprire il tessuto costrutto. Riportare la piastra per l&#39;incubatore (Figura 2). </li></ol> Parte 5: risultati Rappresentante Quando questo protocollo è eseguita correttamente, i mioblasti contenente costruire il tessuto è pronto per l&#39;impianto in vivo (cioè quando viene rimosso dallo stampo) o per ulteriori analisi in vitro dopo 2 giorni. <img src="/files/ftp_upload/1137/Figure1.jpg" alt="" /> Esempi Figura 1. Completato di costruire stampi (vedi punto 1.5). <img src="/files/ftp_upload/1137/Figure2.jpg" alt="" width="400" height="321" /> Figura 2. Un solidificato mioblasti contenente tessuto costruire [1]. <img src="/files/ftp_upload/1137/Figure3.jpg" alt="" /> Figura 3. Sezioni H &amp; E macchiato di un mioblasti contenente tessuto costruire. &quot;A&quot; rappresenta una sezione longitudinale e &quot;B&quot; mostra una sezione trasversale.

<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>

Gli esperimenti sugli animali sono stati eseguiti secondo le linee guida e regolamenti stabiliti dalla cura degli animali e del Comitato Istituzionale uso in bambini Hospital di Boston.

Le forme in cui il costrutto tessuto sarà cast può essere effettuato in qualsiasi forma e dimensione, ma ci deve essere almeno due punti di attacco. In caso contrario, la matrice e le cellule formano una struttura sferica e le cellule muoiono. Nel presente protocollo, si descrive l&#39;utilizzo di una maglia in poliestere per questo scopo, ma abbiamo anche utilizzato con successo in rete in acciaio inox. Ovviamente, più grandi stampi richiederà più celle e un volume maggiore di altri ingredienti. Quando si effettua...

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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

Nonostante il fatto che i pacemaker elettronici sono salvavita dispositivi medici, le loro prestazioni a lungo termine nei pazienti pediatrici può essere problematico a causa delle restrizioni imposte dalle ridotte dimensioni di un bambino e la loro crescita inevitabile. Di conseguenza, vi è una reale necessità di terapie innovative progettate specificamente per i pazienti pediatrici con disturbi del ritmo cardiaco. Proponiamo che una alternativa biologica conduttivo costituito da una matrice a base di collagene contenenti autologously cellule derivate potrebbe adattarsi meglio alla crescita, ridurre la necessità di interventi chirurgici ricorrenti, e migliorare notevolmente la qualità della vita per questi pazienti. Nel presente studio, si descrive una procedura per l&#39;integrazione primaria mioblasti scheletrici colture di cellule in una matrice idrogel di moda un costrutto tessuto chirurgicamente impiantabili che servirà come un condotto elettrico tra le camere superiori e inferiori del cuore. In ultima analisi, possiamo anticipare utilizza questo tipo di ingegneria tessutale per ripristinare atrioventricolare di conduzione elettrica nei bambini con arresto cardiaco completo. In considerazione di questo, abbiamo isolare mioblasti dai muscoli scheletrici di ratti neonati Lewis e la piastra di loro su piatti tessuto laminina rivestite cultura utilizzando una versione modificata di protocolli stabiliti<sup&gt; [2, 3]</sup&gt;. Dopo uno o due giorni, cellule coltivate vengono raccolti e mescolati con gli antibiotici, il collagene di tipo 1, Matrigel ™ e NaHCO<sub&gt; 3</sub&gt;. Il risultato è una viscosa, soluzione uniforme che può essere gettato in uno stampo di quasi ogni forma e dimensione<sup&gt; [1, 4, 5]</sup&gt;. Per il nostro tessuto costruisce, ci avvaliamo di collagene di tipo 1 isolato dalla pelle di agnello del feto utilizzando le procedure standard<sup&gt; [6]</sup&gt;. Una volta che il tessuto è solidificato a 37 ° C, la cultura dei media è attentamente aggiunto alla piastra fino a quando il costrutto è sommerso. L&#39;ingegneria tessutale è poi permesso di condensare ulteriormente attraverso la disidratazione per altri 2 giorni, a quel punto è pronto per<em&gt; In vitro</em&gt; Valutazione o chirurgico-impianto.

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

Fabrication of Myogenic Engineered Tissue Constructs

In questo studio dimostriamo realizzazione di costruzioni a base di collagene dei tessuti, contenenti mioblasti scheletrici. Questi costrutti ingegnerizzati in 3-D possono essere usati per sostituire o riparare i tessuti<em&gt; In vivo</em&gt;. Per i nostri scopi, abbiamo progettato questi come un canale atrioventricolare elettrico per la riparazione di blocco cardiaco completo<sup&gt; [1]</sup&gt;.

Fabbricazione di miogenici Costrutti di ingegneria tessutale

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 Views.  Children's Hospital Boston and Harvard Medical School. In questo studio dimostriamo realizzazione di costruzioni a base di collagene dei tessuti, contenenti mioblasti scheletrici. Questi costrutti ingegnerizzati in 3-D possono essere usati per sostituire o riparare i tessuti In vivo. Per i nostri scopi, abbiamo progettato questi come un canale atrioventricolare elettrico per la riparazione di blocco cardiaco completo [1].

Video: Fabrication of Myogenic Engineered Tissue Constructs

PDMS Device Fabrication and Surface Modification

PDMS fabbricazione del dispositivo e la modifica della superficie

Ricerca

Biologia

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.

Fabbricazione dei canali termoplastico Microfluidic

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-sedimentazione di analisi, per l&#39;Analisi del Binding Protein a 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.

Fabbricazione di elettrodi amperometrici

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.

L&#39;impianto di ingegneria tessutale nel Cuore 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.

Crioconservazione del mouse Sperm and Recovery utilizzando il Kit · I Cryo

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 costrutti omologhi ricombinazione<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.

Preparazione di primaria Miogenica precursori cellulari / Culture mioblasti dal basale vertebrati Lignaggi

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.

Fago-mediata consegna di mirata sRNA Costruisce per abbattere l&#39;espressione genica in<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.

La dissezione del Auditory Bulla nei topi postnatale: Isolamento delle ossa dell&#39;orecchio medio e l&#39;analisi istologica

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 ...