Este trabalho foi apoiado pelo National Institutes of Health concede NS047726, NS072027, e AR052646.

Os métodos neste estudo foram realizados de acordo com a ética Feinberg School of Medicine da Northwestern University Animal Care Institucional e Comitê de Uso (IACUC) aprovou diretrizes. Foram feitos todos os esforços para minimizar o sofrimento. 1. Processo Experimental para in vivo Eletroporação do brevis flexor (FDB) Bundle Muscle no mouse <ol><li> Plasmídeos de desenho para expressão in vivo utilizando um promotor conhecido para expressar em células de mamífero (por exemplo, citomegalovírus, CMV) ou no músculo (por exemplo, músculo de creatina quinase, MCK) 6,7. Plasmídeos maiores podem expressar em níveis mais baixos. O promotor CMV foi usado extensivamente 6,7. </li><li> Purifica-se plasmídeos a partir de E. coli utilizando endotoxina condições livres por as instruções do fabricante. Dissolve-se o plasmídeo em estéril, livre de endotoxinas tampão TE a 2 - 5 ug de plasmídeo / ul. </li><li> Dilui-se o plasmídeo e aliquotar para um ConcentraçãN com 20 ug de ADN em 10 ul de TE estéril isento de endotoxina (2 ug / uL) em tampão por injecção um tubo de microcentrífuga estéril. Prepare uma aliquota por injecção. </li><li> Diluir o stock de hialuronidase para 1x com solução salina estéril tamponada com fosfato sem cálcio e magnésio (PBS - / -), dando uma concentração final de 8 unidades. Alíquota de 10 ul de hialuronidase diluído por injecção num tubo de microcentrífuga estéril. </li><li> Anestesiar o rato usando 2,5% de isoflurano até sedado e não responde a cauda ou beliscar dedo do pé. Colocar o animal numa posição supina sobre uma almofada de aquecimento ou mesa de aquecimento para manter a temperatura corporal normal. </li><li> Limpe a pata do rato com uma aplicação alternada de um matagal iodopovidona e álcool três vezes, usando uma técnica de preparação originada pela instalação agulha e irradiando para fora. Esta técnica mantém o site agulha em uma condição asséptica, minimizando introdução de agentes patogénicos. </li><li> Injectar a pata de the rato com 10 ul da solução 1x hialuronidase (2 mg / ml = 8 unidades) entre a pele e o músculo utilizando uma seringa esterilizada de 1 ml. Hialuronidase digere componentes da matriz extracelular para permitir a entrada mais eficiente do plasmídeo em miofibras. Introduzir na base do calcanhar e promover a agulha na direcção dos dedos do pé. Libertam lentamente o líquido quando a agulha é retraída. O local de injecção sob a pele vai se expandir e começar a virar rosa. </li><li> Repita os passos 1.6 e 1.7 no pé contralateral. </li><li> Desligue a anestesia e coloque o mouse de volta na gaiola. Permitir que o mouse para se recuperar totalmente da anestesia. </li><li> Espere 2 horas. </li><li> Repita os procedimentos de esterilização anestesiar e do pé, os passos 1.5 e 1.6. </li><li> Injectar o ADN plasmídico diluído na almofada da pata da mesma forma como a hialuronidase. Verifique se o volume máximo é de 20 l ou menos. Para plasmídeo de expressão dupla injectar 20 ug de cada plasmídeo com um volume máximo de 20 &amp;# 181; l ou menos. </li><li> Repetir a injecção do plasmídeo em planta da pata contralateral ou usar o pé contralateral para injecções de controlo. </li><li> No pé que foi inicialmente injectado, levantar a pele para longe do músculo sublinhado com uma pinça fina e colocar uma agulha G 27 com as bolas de pele perto dos dedos numa posição perpendicular à linha de curar-dedo do pé. Tome um segundo estéril 27 G agulha e colocá-lo horizontalmente através da pele no calcanhar. Coloque agulhas paralelas entre si ~ 1 cm de distância. </li><li> Usando grampos jacaré, eletrodos de fixação para as agulhas paralelas certificando-se de que as agulhas não entre em contato com o outro. O uso de massa de modelar pode proteger os grampos no lugar. </li><li> Conectar eléctrodos de um estimulador eléctrico. </li><li> Electroporate o músculo através da aplicação de 20 pulsos, 20 ms de duração / cada, em 1 Hz, a 100 V / cm. Os dedos podem se contrair com a estimulação. </li><li> Repetir o procedimento de estimulação no pé contralateral, se necessário. </li><li>Remover agulhas. Limpe a pata com iodopovidona matagal. </li><li> Desligue a anestesia. Devolver o mouse para a gaiola de recuperação e atividade monitor. Se o processo foi conduzido como se esperava, o animal deve recuperar a deambulação normal dentro de 30 minutos e não exibem nenhuma diferença no nível de actividade de locomoção ou comportamento pré-injecção. Portanto, não há necessidade de analgésicos pós-procedimento. Após a recuperação, devolver o animal para o viveiro. Nota: A expressão da proteína pode ser ensaiada tão cedo quanto 48 horas após a electroporação. No entanto, para permitir a recuperação mais completa após a electroporação, nós preferimos o isolamento e estudo de miofibras 7 - 14 dias após a electroporação 10. A expressão pode ser ensaiada tanto tempo quanto 4 semanas após a electroporação. </li></ol> 2. Procedimento Experimental para o isolamento do brevis flexor (FDB) Fibers <ol><li> Colagenase Thaw. Para cada pacote FDB preparar dois poços em uma placa de 12 poços de cultura de tecidos. Numa bem adicionar 1 ml de pré-aquecido Eagles Modificado por Dulbecco Meios mais albumina de soro bovino (DMEM + BSA) e na outra bem adicionar 1 ml de pré-aquecido Solução de Ringer. Além disso, adicione frescos 10 ml de solução de Ringer 1x aos 35 mm SYLGARD prato. </li><li> Sacrificar o animal através de CO 2 ou inalação de gás anestésico, seguido por deslocamento cervical ou outro método para garantir a morte. </li><li> Retire os pés traseiros acima da articulação do tornozelo utilizando uma lâmina de barbear limpa e mergulhe na solução de Ringer 1x nos 35 mm SYLGARD prato. </li><li> Pin um pé apertado, único virado para cima, para o prato Sylgard com 30 g agulhas através de cada uma das 5 pontas do dedo do pé e no tornozelo. </li><li> Começando no tornozelo, corte a pele para cima o lado do pé ao longo da linha do cabelo para os dedos dos pés, cuidado para não nick o músculo sob a pele. Repita no outro lado do pé. </li><li> Segurando a pele na base do tornozelo com uma pinça fina, cortar a pele do outro lado do calcanhar. </li><li> Levantar a pelebater com uma pinça, apontar sua tesoura para a pele, para não nick o músculo. Cortar o tecido conjuntivo subjacente, removendo eficazmente a pele. </li><li> Para remover o feixe muscular FDB cortar o grande tendão no calcanhar para libertar o tendão a partir do osso. Segurando o tendão com uma pinça, disseque o pacote FDB do tendão grande branco cuidadosamente remover qualquer tecido conjuntivo anexado ao FDB com uma tesoura. Se feito corretamente o pacote será fácil decolar dos tendões subjacentes revelando tendões brancas brilhantes que conduzem aos dígitos individuais. Corte o FDB nos tendões perto dos dedos liberando o pacote do pé. </li><li> Coloque todo o feixe muscular no poço contendo DMEM + BSA. </li><li> Repita com o pé contralateral. </li><li> Uma vez que todos os músculos FDB são isolados, adicionar 100 ul de colagenase a cada poço contendo DMEM + BSA e músculo. </li><li> Verificar o sucesso de electroporação num microscópio de fluorescência invertido antes da digestão. Se properl feitoy, para cima de 90% de eficiência de transfecção pode ser alcançado. </li><li> Incubar a placa numa atmosfera humidificada, a 37 ° C na incubadora de CO2 a 10% durante cerca de 1 h. O tempo de incubação pode variar, dependendo do modelo da doença e do fundo e o lote de colagenase. </li><li> Após 1 hora, transferir cuidadosamente o pacote FDB para a solução de Ringer bem contendo somente. </li><li> Tritura-se o feixe de FDB no poço com a solução de Ringer, utilizando uma pipeta de 1 ml. Cortar a extremidade da ponta da pipeta com uma lâmina de barbear limpa de tal forma que o pacote pode facilmente passar através da ponta da pipeta. Gentilmente triturar o músculo (~ 15 vezes) permitindo que o pacote para passar cima e para baixo paralela à ponta. Fibras intactas vai cair no Ringers Solution. Quando as fibras não são facilmente cair fora do pacote, coloque o músculo volta para solução de colagenase. </li><li> Placa das fibras isoladas no prato ~ 500 mL por placa. </li><li> Permitir que o músculo para digerir no poço durante 15 minutos. </li><li> Repita os passos de 2.15para 2,17 até que o feixe diminui em tamanho e a maioria das fibras está dissociado do feixe (geralmente um total de 2-3 vezes). Uma vez que o músculo facilmente dissociado passa através de uma ponta de pipeta de 1 ml. </li><li> Vamos fibras anexar ao prato para um mínimo de 15 min. Solução de Ringer fresca pode ser adicionado à placa para diluir colagenase residual ou alterada dependendo das limitações de tempo. Nota: As fibras estão agora prontos para a imagem latente. </li></ol> 3. Procedimento Experimental para a visualização da membrana induzida por laser danos usando microscopia confocal <ol><li> Fibras de carga imediatamente antes da imagiologia com 300 ul de 2,5 mM FM 1-43 de FM 4-64 corante em solução de Ringer. Geralmente, as fibras de imagem de 15 min a 2 horas após o seu isolamento. No entanto, as fibras podem ser visualizados até 24 horas após o isolamento. </li><li> Coloque o prato com fundo de vidro em um microscópio confocal equipado com um laser UV e um objetivo 60X / 63X. </li><li> Localize uma fibra. Certifique-se a fibra está firmemente ligadopara o prato tocando a base de microscópio. Excluir fibras que foram danificados durante o processo de dissociação. Fibras danificadas podem conter bolhas de membrana, altos níveis de captação do corante FM, onda ou dobrar intensamente. </li><li> Para induzir danos na membrana da fibra, o sarcolema posicionar no centro da janela de imagem com um campo claro de núcleos. Concentre-se no sarcolemma utilizando o canal de FM corante tal que a sarcolemma é extremamente afiada. </li><li> Coloque o cabelo ROI cruz no sarcolema utilizando o canal de FM 4-64. </li><li> Irradiar um ponto 1 pixel a 80% de potência por 3 segundos (~ 350 nm x 350 nm área). Corante FM irá entrar na célula, no local da lesão. Potência e tempo pode ser ajustada para aumentar ou diminuir a área de insulto. </li><li> Captar imagens da fibra antes do dano, no momento do dano, a cada 2 s de pós-dano, em seguida, a cada 10 segundos até 5 minutos. Tempo pode ser ajustado, dependendo da necessidade. Imagens individuais podem ser convertidos em filmes de lapso de tempo em Image J. </li><li> Repita os passos3.3 a 3.7 em até 3 fibras por prato. </li></ol>

<ol>
	<li>Blau, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+the+differentiated+state.">Plasticity of the differentiated state.</a> Science. 230, 758-766 (1985).</li><li>Yaffe, D., Saxel, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+passaging+and+differentiation+of+myogenic+cells+isolated+from+dystrophic+mouse+muscle.">Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle.</a> Nature. 270, 725-727 (1977).</li><li>Richler, C., Yaffe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vitro+cultivation+and+differentiation+capacities+of+myogenic+cell+lines.">The in vitro cultivation and differentiation capacities of myogenic cell lines.</a> Dev Biol. 23, 1-22 (1970).</li><li>Yaffe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retention+of+differentiation+potentialities+during+prolonged+cultivation+of+myogenic+cells.">Retention of differentiation potentialities during prolonged cultivation of myogenic cells.</a> Proc Natl Acad Sci USA. 61, 477-483 (1968).</li><li>Mamchaoui, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalized+pathological+human+myoblasts:+towards+a+universal+tool+for+the+study+of+neuromuscular+disorders.">Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders.</a> Skelet muscle. 1, 34 (2011).</li><li>DiFranco, M., Quinonez, M., Capote, J., Vergara, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+transfection+of+mammalian+skeletal+muscles+using+in+vivo+electroporation.">DNA transfection of mammalian skeletal muscles using in vivo electroporation.</a> J Vis Exp: JoVE. , (2009).</li><li>Swaggart, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annexin+A6+modifies+muscular+dystrophy+by+mediating+sarcolemmal+repair.">Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair.</a> Proc Natl Acad Sci USA. 111, 6004-6009 (2014).</li><li>Kerr, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysferlin+stabilizes+stress-induced+Ca2++signaling+in+the+transverse+tubule+membrane.">Dysferlin stabilizes stress-induced Ca2+ signaling in the transverse tubule membrane.</a> Proc Natl Acad Sci USA. 110, 20831-20836 (2013).</li><li>Anderson, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+micropeptide+encoded+by+a+putative+long+noncoding+RNA+regulates+muscle+performance.">A micropeptide encoded by a putative long noncoding RNA regulates muscle performance.</a> Cell. 160, 595-606 (2015).</li><li>Roche, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+and+histological+changes+in+skeletal+muscle+following+in+vivo+gene+transfer+by+electroporation.">Physiological and histological changes in skeletal muscle following in vivo gene transfer by electroporation.</a> Am J Physiol Cell Physiol. 301, C1239-1250 (2011).</li><li>Reddy, A., Caler, E. V., Andrews, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+membrane+repair+is+mediated+by+Ca(2+)-regulated+exocytosis+of+lysosomes.">Plasma membrane repair is mediated by Ca(2+)-regulated exocytosis of lysosomes.</a> Cell. 106, 157-169 (2001).</li><li>Wang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+blebbing+as+an+assessment+of+functional+rescue+of+dysferlin-deficient+human+myotubes+via+nonsense+suppression.">Membrane blebbing as an assessment of functional rescue of dysferlin-deficient human myotubes via nonsense suppression.</a> J Appl physiol. 109, 901-905 (2010).</li><li>Cai, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MG53+nucleates+assembly+of+cell+membrane+repair+machinery.">MG53 nucleates assembly of cell membrane repair machinery.</a> Nat Cell Biol. 11, 56-64 (2009).</li><li>Marg, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sarcolemmal+repair+is+a+slow+process+and+includes+EHD2.">Sarcolemmal repair is a slow process and includes EHD2.</a> Traffic. 13, 1286-1294 (2012).</li><li>Bansal, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+membrane+repair+in+dysferlin-deficient+muscular+dystrophy.">Defective membrane repair in dysferlin-deficient muscular dystrophy.</a> Nature. 423, 168-172 (2003).</li><li>Defour, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysferlin+regulates+cell+membrane+repair+by+facilitating+injury-triggered+acid+sphingomyelinase+secretion.">Dysferlin regulates cell membrane repair by facilitating injury-triggered acid sphingomyelinase secretion.</a> Cell Death Dis. 5, e1306 (2014).</li></ol>

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O uso de electroporação para estudar as proteínas marcadas por fluorescência in vivo, é idealmente adequado para o estudo do músculo devido à ausência de modelos de linhas celulares adequadas que reproduzem com fidelidade a estrutura da célula de músculo inteira. Este protocolo descreve a utilização de electroporação e microscopia confocal de alta resolução para fornecer imagens detalhadas de localização de proteínas e translocação após o ferimento a laser. Este método pode ser adaptado p...

Myofibers individuais são células sinciciais grandes, altamente organizados. Existem alguns modelos de cultura celular para o músculo; No entanto, estes modelos têm como principal limitação que não diferenciar plenamente em myofibers maduros. Por exemplo, as linhas de células C2C12 e L6 são derivados de ratinhos e ratos, respectivamente 1-4. Sob condições de privação de soro, as células mononucleares &quot;mioblastos-like&quot; cessar proliferação, submetido a retirada do ciclo celular, e entrar no programa miogênica formando células multinucleadas com sarcômeros, referido como myotubes. Com condições de cultura prolongados, myotubes podem exibir propriedades contráteis e &quot;contração&quot; na cultura. Linhas de células humanas têm também sido agora estabelecido 5. Em adição a estas linhas celulares imortalizadas, mioblastos mononucleares podem ser isoladas a partir do músculo e sob as mesmas condições de privação de soro irá formar miotubos. Estas linhas celulares e culturas primárias de mioblastos são altamente usarful porque eles podem ser transf ectadas com plasmídeos ou transduzidas com vírus e utilizado para estudar processos celulares biológicas básicas. No entanto, essas células, mesmo quando induzidos a formar miotubos, não têm muitas das características marcantes da organização músculo maduro. Especificamente, miotubos são muito menores do que miofibras maduras individuais e não têm a forma normal de miofibras. Criticamente, myotubes falta transversais (T-) túbulos, a rede membranoso necessária para eficiente liberação de Ca2 + em todo o myoplasm. Um método alternativo para mioblastos primários ou linhas de células miogénicas implica a utilização de miofibras maduras. Transgénese pode ser utilizada para determinar a expressão de proteínas marcadas, mas este método é dispendioso e demorado. Electroporação in vivo de músculo do rato tem surgido como um método preferido para a sua velocidade e fiabilidade 6-10. Métodos para electroporação in vivo e o isolamento eficiente de miofibras have foi otimizado para o flexor digitorum brevis rato (FDB) 6. Os métodos podem ser prontamente concluída e são minimamente invasiva para induzir a expressão in vivo a partir de plasmídeos. Esta abordagem é agora combinado com métodos de imagem de alta resolução de imagem, incluindo após a interrupção do laser do sarcolema 6,7. A combinação de corantes fluorescentes e de expressão de proteínas marcado por fluorescência pode ser utilizado para monitorar os processos biológicos das células em miofibras maduras.

<table>
	<tbody>
		<tr>
			<td>DMEM+A2:D32D42A2:D31AA2:D29</td>
			<td>Life Technologies</td>
			<td>11995-073</td>
			<td>Dissection Media 
			Dilution: 50ml +100g BSA</td>
		</tr>
		<tr>
			<td>Collagenase, Type II</td>
			<td>Life Technologies</td>
			<td>17101-015</td>
			<td>Fiber Digestion 
			Dilution: 160mg / 4ml DMEM (stock 40mg/ml aliquot)</td>
		</tr>
		<tr>
			<td>Falcon 12-well dishes</td>
			<td>Fisher Scientific</td>
			<td>877229</td>
			<td>Fiber Digestion</td>
		</tr>
		<tr>
			<td>Ringers Solution</td>
			<td></td>
			<td></td>
			<td>Fiber Digestion and Imaging 
			Dilution: 146 mM NaCl 
			5 mM&#160;&#160; KCl 
			2 mM&#160;&#160; CaCl2 
			1 mM&#160;&#160; MCl2 
			10mM HEPES 
			pH7.4 in H2O</td>
		</tr>
		<tr>
			<td>1ml Pipettes</td>
			<td>Axygen</td>
			<td>T-1000-C-R</td>
			<td>Digestion</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Razor Blades</td>
			<td>Personna</td>
			<td>94-120-71</td>
			<td>Digestion</td>
		</tr>
		<tr>
			<td>Precision Glide 30G needle</td>
			<td>BD Biosciences</td>
			<td>305128</td>
			<td>Dissection</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>1x PBS (-/-)</td>
			<td>Life Technologies</td>
			<td>14190-250</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Sylgard 184</td>
			<td>Fisher Scientific</td>
			<td>50-366-794</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>FST by Dumont</td>
			<td>#5/45</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Roboz</td>
			<td>RS-4913</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>World Precision Instruments</td>
			<td>500260</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td>Dissection media 
			Dilution: 100mg / 50ml DMEM</td>
		</tr>
		<tr>
			<td>Falcon 60mm dishes</td>
			<td>Fisher Scientific</td>
			<td>08772B</td>
			<td>Dissection- used to hold sylgard</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Clay</td>
			<td></td>
			<td></td>
			<td>Electroporation</td>
		</tr>
		<tr>
			<td>Stimulator&#160;</td>
			<td>Grass</td>
			<td>s88x</td>
			<td>Electroporation</td>
		</tr>
		<tr>
			<td>Clamps</td>
			<td>Radio Shack</td>
			<td>270-356</td>
			<td>Electroporation</td>
		</tr>
		<tr>
			<td>Triadine</td>
			<td>Aplicare</td>
			<td>82-220</td>
			<td>Electroporation</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Precision Glide 27G needle</td>
			<td>BD Biosciences</td>
			<td>305136</td>
			<td>Electroporation</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Simulator</td>
			<td>Grass</td>
			<td>SIU-V</td>
			<td>Electroporation</td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Covidien</td>
			<td>2146</td>
			<td>Electroporation</td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma</td>
			<td>H4272</td>
			<td>Injection 
			Dilution: Add 160ul PBS (1X) to 40&#181;l 5x stock</td>
		</tr>
		<tr>
			<td>1cc U-100 Insulin Syringe (28G1/2)&#160;</td>
			<td>BD Biosciences</td>
			<td>329420</td>
			<td>Injection</td>
		</tr>
		<tr>
			<td>Eppendorf Tubes</td>
			<td>Fisher Scientific</td>
			<td>02-682-550</td>
			<td>Injection</td>
		</tr>
		<tr>
			<td>35mm glass bottom Microwell dish</td>
			<td>MaTek</td>
			<td>P35G-1.5-14-C</td>
			<td>Microscopy</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>FM 4-64</td>
			<td>Life Technologies</td>
			<td>T-13320</td>
			<td>Microscopy 
			Dilution: Final 2.5 ug/ml</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>FM 1-43</td>
			<td>Life Technologies</td>
			<td>T-35356</td>
			<td>Microscopy 
			Dilution: Final 2.5 ug/ml</td>
		</tr>
		<tr>
			<td>Endotoxinfree Plasmid Maxi Kit</td>
			<td>Qiagen</td>
			<td>12362</td>
			<td>Plasmid Purification</td>
		</tr>
		<tr>
		</tr>
	</tbody>
</table>

dna electroporation, isolation and imaging of myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

A electroporação é uma técnica minimamente invasiva que introduz eficientemente DNA de plasmídeo purificado no brevis flexor (FDB) feixe muscular (Figura 1A). Sete dias após a transfecção proteína marcada fluorescentemente é visualizado em miofibras isolados (Figura 1B). Isolado fibras são então colocadas em placas e preparado para imagiologia de um microscópio confocal. As fibras podem ser submetidas a uma lesão induzida por laser. Corante...

Watch this Scientific Journal Video about DNA Electroporation, Isolation and Imaging of Myofibers at JoVE.com

DNA Electroporation, Isolation and Imaging of Myofibers

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

DNA Eletroporação, Isolamento e imagem das miofibras

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

dna-electroporation-isolation-and-imaging-of-myofibers

Research

JoVE Journal

Biology

12.2K visualizações.  Northwestern University. This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure. 

Vídeo: DNA Electroporation, Isolation and Imaging of Myofibers

Isolation of Genomic DNA from Mouse Tails

Isolamento de DNA genômico de Tails rato

Biologia

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

Eletroporação de Micobactérias

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

Transfecção de DNA de mamíferos usando músculos esqueléticos In Vivo Eletroporação

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

Duas e três dimensões imagens de células vivas de Proteínas de DNA resposta a danos

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Isolamento e Cultura de miofibras individuais e suas células satélites do músculo esquelético

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

Análise de Desenvolvimento Embrionário e larval Zebrafish esqueléticos miofibras de Preparações dissociadas

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

Combinando uma única molécula de Manipulação e imagem para o estudo das interações proteína-DNA

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

Imagens em tempo real de uma única Engineered RNA transcritos em células vivas usando raciométrica Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

De gravidade reduzida Manifestações ambiente de hardware de uma tecnologia de mistura de fluxo Cytometer e Companion microfluídicos Prototype Miniaturized

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Amplificação, sequenciamento de próxima geração, e Genomic DNA Mapeamento das retrovirais Integração Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...