Este trabalho é suportado por verbas de pesquisa dos Institutos Nacionais de Saúde (HL068915; HL088206), o Prêmio Pesquisador Nova do Fundo de Investigação Thrasher, e contribuições para o Fundo de Condução Cardíaca do Hospital Infantil de Boston.

Parte 1: Montar construir moldes de fundição <ol><li> Use uma lâmina de barbear para cortar pela metade tubos de silicone (VWR) e corte-o em três pedaços cm de comprimento. </li><li> Coloque uma gota de implante de silicone RTV-grade adesiva (Rhodia) no interior de cada extremidade do tubo. </li><li> Colocar rapidamente um pequeno pedaço (1 cm x 1 cm) de poliéster malha (McMaster-Carr) na queda adesivo de silicone e alinhá-lo com a extremidade do tubo. Isso irá proporcionar uma superfície ligeiramente elevada e plana para fixação construir. Repita o procedimento para a outra extremidade. </li><li> Permitir que o molde para secar em temperatura ambiente por 3 dias. É útil para fazer 20-30 moldes ao mesmo tempo, uma vez que estas são essencialmente descartáveis. </li><li> Uma vez que o adesivo é completamente seco, armazene esses moldes num copo cheio de etanol 70% até que sejam necessários. Este passo ajudará a esterilizar os moldes eo produto final é mostrado na Figura 1. </li></ol> Parte 2: Preparação para o isolamento de células mioblastos <ol><li> Diluir 1 mg laminina (Sigma) em 250 mL 0,22 M esterilizada por filtração, tampão fosfato (PBS) contendo 1% de penicilina / estreptomicina (Invitrogen) e Fungizone 1% (Invitrogen). </li><li> Casaco 150 milímetros placas de cultura de tecidos (BD Falcon) com 4 mL da solução diluída de laminina passo 1.1 e incubar a 37 º C por pelo menos 4 horas antes do plaqueamento os mioblastos esqueléticos primários. </li><li> Tornar o meio de mioblastos, misturando F-10 Ham Mistura de nutrientes (Sigma) com FBS 20% (Atlanta Biologicals), 5 ng / L Fator de crescimento básico de fibroblastos (Promega), 1% Penicilina / Estreptomicina (Invitrogen), e Fungizone 1% ( Invitrogen). </li><li> Preparar a solução de digestão do músculo esquelético, dissolvendo 1 g de ~ 295 U / mg de colagenase 2 (Worthington) em 100 mL 2,4 U / mL Dispase-2 (Roche). Adicionar 0,037 g CaCl 2 e misture bem. Filtro de esterilizar o tampão de digestão usando um disco de 0,2 mM de filtro montado em uma seringa de 10 mL e alíquotas armazenar a -20 ° C. Coloque a quantidade necessária para a digestão do tecido a 37 ° C, juntamente com a mídia mioblastos. </li></ol> Parte 3: isolamento mioblastos de músculo esquelético <ol><li> Utilizando uma pinça e uma tesoura pequena, retire músculos paravertebrais de ratos mortos neonatal Lewis por corte nas costas ao longo da medula espinhal, retirar as camadas da pele e fáscia e, em seguida, extirpando os músculos. </li><li> Em uma capa de cultura de tecidos, coloque os músculos excisadas em um tubo de 50 ml (BD Falcon) preenchida com 40 mL de solução de sal 1X Hanks Balanced (HBSS) (Invitrogen) no gelo. Permita que as tiras do músculo em repouso para a parte inferior do tubo e retire cuidadosamente a maior parte dos restos de líquido e sangue por sucção a vácuo com uma pipeta Pasteur estéril em uma capa de cultura (Baker). </li><li> Despeje as peças muscular colhidas em uma placa de cultura 150 mm e remover o máximo do líquido restante possível com sucção. Usando duas lâminas de barbear única gumes mince o tecido para uma pasta grossa. Despeje pré-aquecido solução digestão sobre a pasta e transferir a mistura para um tubo mL fresco 50 usando uma pipeta 10 mL. Durante a digestão enzimática, o tecido é ocasionalmente triturado (sem bolhas) com uma pipeta de 10 mL provido em um fio Pipetar-Aid (Drummond). Mais uma vez, permitir que o tecido para resolver e remover o excesso de líquido. </li><li> Colocar o tubo em uma plataforma de balanço e digerir a 37 º C por cerca de 30 min. </li><li> Uma vez que o músculo é liquefeito, passar a solução através de um filtro de células 70 mM (BD Falcon) e centrifugar a temperatura ambiente por 10 min a 600 xg (Beckman GP). Com uma pipeta Pasteur e sucção, remover o máximo de líquido possível e ressuspender o sedimento com 10 ml de meio de mioblastos quente. </li><li> Pré-plate as células em um prato de 150 milímetros de cultura de tecidos revestidos por 15 minutos para ajudar a remover fibroblastos. </li><li> Transferência das células que ainda não anexado ao cultureware laminina-revestido e diluir para que haja uma placa para cada 1 a 2 filhotes de ratos. Coloque a placa em um conjunto incubadora umidificada a 37 ° C com 5% de CO 2. </li></ol> Parte 4: Fundição construções da engenharia de tecidos (5 mL) <ol><li> Após 1 a 2 dias, retire 10 da 150 milímetros mioblastos placas de cultura de tecidos da incubadora, lave-os duas vezes com 37 ° C 1X PBS, e adicionar 4 mL de morno 0,05% Tripsina-EDTA para cada prato. Retornar as placas para a incubadora por 5 min. </li><li> Colher os mioblastos destacada das placas no bairro cultura e recolher o líquido em um tubo de 50 mL (BD Falcon). Use 10 mL de meio de mioblastos para lavar todos os pratos e combinar isso com as células no tubo de ensaio. Agregar as células em temperatura ambiente por 10 min a 600 xg (Beckman GP) e remova o excesso de líquido, tal como descrito no passo 3.5. </li><li> Ressuspender as células mioblastos em 1,6 mL de meio de mioblastos e colocar no gelo. </li><li> Usando pinça, retire cuidadosamente quatro moldes construir a partir do passo 1.5 e lave-os duas vezes com PBS 1X. Sucção a vácuo off todos os vestígios de PBS com uma pipeta Pasteur, antes de colocar cada molde em um cu bem 6-tecidoLTURE placa (BD Falcon). </li><li> Reúna os outros reagentes necessários para a preparação de construir (F10 Ham 10X é, antibióticos, colágeno tipo 1 [3,9 mg / mL] [6] e Matrigel ™) sobre o gelo e manter meios de comunicação mioblastos a 37 º C. Combine 500 mL 10X Ham F10, 100 mL penicilina / estreptomicina, 100 Fungizone mL, 1,6 mL de colágeno, e 750 ™ Matrigel mL em tubo cônico de 15 mL. Misturar a solução por 10 segundos usando um Mini-Vortexer (VWR) definida para 10 e colocar no gelo. </li><li> Em seguida, adicione 80 mL de NaHCO 3 ea suspensão mioblastos a partir do passo 3.3 para a mistura a partir do passo 3.5. Vortex toda a mistura por 10 segundos e retornar o tubo para o balde de gelo para um poucos minutos para limpar a solução de bolhas. </li><li> Cuidadosamente elenco a mistura nos moldes antes que comece a solidificar usando uma pipeta 10 mL. Cada molde irá realizar cerca de 1 mL do líquido viscoso. Tente evitar a introdução de bolhas de ar durante este procedimento. </li><li> Transferir cuidadosamente a placa contendo as construções elenco para a incubadora. Depois de ~ 30 min, retire as construções solidificadas da incubadora e adicione cuidadosamente médio mioblastos a cada poço para cobrir o tecido construir. Devolver a placa para a incubadora (Figura 2). </li></ol> Parte 5: Os resultados representativos Quando este protocolo é executado corretamente, o construto de tecido contendo mioblastos está pronto para implantação em vivo (ou seja, quando removido do molde) ou para mais nas análises in vitro após 2 dias. <img src="/files/ftp_upload/1137/Figure1.jpg" alt="" /> Figura 1. Exemplos de concluídos moldes construir (veja o Passo 1.5). <img src="/files/ftp_upload/1137/Figure2.jpg" alt="" width="400" height="321" /> Figura 2. A solidificou mioblastos contendo tecido construir [1]. <img src="/files/ftp_upload/1137/Figure3.jpg" alt="" /> Figura 3. H &amp; E cortes corados de um tecido mioblastos contendo construir. &quot;A&quot; mostra uma seção longitudinal e &quot;B&quot; mostra um corte 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>

Experimentos em animais foram realizados de acordo com as diretrizes e regulamentos estabelecidos pela Institutional Animal Care e do Comitê Use do Hospital Infantil de Boston.

Os moldes em que a construção de tecido serão lançados podem ser feitas em qualquer formato e tamanho, no entanto, é preciso haver pelo menos dois pontos de fixação. Caso contrário, a matriz e as células formam uma estrutura esférica e as células morrem. No protocolo, descrevemos o uso de uma malha de poliéster para esta finalidade, mas também temos utilizado com sucesso malha de aço inoxidável. Obviamente, moldes maiores exigirá mais células e um maior volume de outros ingredientes. Ao fazer os moldes,...

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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

Apesar do facto de pacemakers eletrônicos são salva-vidas dispositivos médicos, o seu desempenho a longo prazo em pacientes pediátricos pode ser problemático, devido a restrições impostas pelo pequeno tamanho de uma criança e seu crescimento inevitável. Conseqüentemente, há uma necessidade genuína para terapias inovadoras projetadas especificamente para pacientes pediátricos com distúrbios do ritmo cardíaco. Propomos que uma alternativa condutora biológica consiste de uma matriz à base de colágeno contendo autologously células derivadas poderiam se adaptar melhor ao crescimento, reduzir a necessidade de cirurgias recorrentes, e melhorar significativamente a qualidade de vida para esses pacientes. No presente estudo, descrevemos um procedimento para a incorporação de culturas de células primárias de mioblastos esqueléticos dentro de uma matriz de hidrogel para moldar uma estrutura de tecido cirurgicamente implantável, que servirá como um conduíte elétrico entre as câmaras superior e inferior do coração. Em última instância, nós antecipamos usando este tipo de engenharia de tecidos para restaurar a condução elétrica atrioventricular em crianças com bloqueio cardíaco completo. Em vista disso, isolamos mioblastos de músculo esquelético de ratos Lewis neonatal e placa-los para laminina revestido pratos de cultura de tecidos usando uma versão modificada de protocolos estabelecidos<sup&gt; [2, 3]</sup&gt;. Depois de 1-2 dias, as células cultivadas são coletados e misturados com os antibióticos, o colágeno tipo 1, Matrigel ™, e NaHCO<sub&gt; 3</sub&gt;. O resultado é uma solução viscosa uniforme que pode ser convertido em um molde de quase qualquer formato e tamanho<sup&gt; [1, 4, 5]</sup&gt;. Para construções nosso tecido, que empregam colágeno tipo 1 isolado da pele de cordeiro fetal usando procedimentos padrão<sup&gt; [6]</sup&gt;. Uma vez que o tecido tem solidificado a 37 ° C, cultura da mídia é cuidadosamente adicionada à placa até que a construção está submerso. A engenharia de tecidos é permitido então condensar ainda mais através de desidratação por mais 2 dias, altura em que ele está pronto para<em&gt; In vitro</em&gt; Avaliação ou cirúrgico-implantação.

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

Fabrication of Myogenic Engineered Tissue Constructs

Aqui, demonstramos a fabricação de colágeno baseado em construções de tecido contendo os mioblastos esqueléticos. Essas construções 3-D de engenharia podem ser usados ​​para substituir ou reparar tecidos<em&gt; In vivo</em&gt;. Para os nossos propósitos, nós projetamos estes como um canal atrioventricular elétrica para a reparação de bloqueio cardíaco completo<sup&gt; [1]</sup&gt;.

Fabricação de Miogênica Constrói engenharia de tecidos

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 visualizações.  Children's Hospital Boston and Harvard Medical School. Aqui, demonstramos a fabricação de colágeno baseado em construções de tecido contendo os mioblastos esqueléticos. Essas construções 3-D de engenharia podem ser usados ​​para substituir ou reparar tecidos In vivo. Para os nossos propósitos, nós projetamos estes como um canal atrioventricular elétrica para a reparação de bloqueio cardíaco completo [1].

Vídeo: Fabrication of Myogenic Engineered Tissue Constructs

PDMS Device Fabrication and Surface Modification

PDMS Fabricação de dispositivos e modificação da superfície

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.

Fabricação dos canais microfluídicos Termoplásticos

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 Ensaio Co Sedimentação; para a Análise da proteína de ligação 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.

Fabricação de eletrodos amperométrica

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.

Implantação de engenharia de tecidos no coração de ratos

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.

Criopreservação do mouse Sperm and Recovery utilizando o 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 Construções recombinação homóloga em<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.

Preparação de Primário Miogênica de Células Precursoras / Culturas de mioblastos de Vertebrados basais Linhagens

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.

Entrega mediada por fago de sRNA alvejado Constrói para bater para baixo Expressão Gênica em<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.

Dissecção da Auditivo Bulla em ratos pós-natais: Isolamento dos ossos do ouvido médio e da análise histológica

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 ...

Methods Article

Fabricação de Miogênica Constrói engenharia de tecidos