Ce travail a été soutenu par les Instituts nationaux de la Santé accorde NS047726, NS072027 et AR052646.

Les méthodes de cette étude ont été effectuées en conformité avec l&#39;éthique Feinberg School of Medicine de la Northwestern University Institutional Animal Care et l&#39;utilisation Comité (IACUC) des lignes directrices approuvées. Tous les efforts ont été faits pour minimiser la souffrance. 1. Procédure expérimentale dans électroporation in vivo de la court fléchisseur des orteils (FDB) Muscle Bundle souris <ol><li> Plasmides de conception pour l&#39;expression in vivo en utilisant un promoteur connu pour exprimer dans des cellules de mammifères (par exemple, le cytomégalovirus, CMV) ou dans le muscle (par exemple, Muscle Créatine kinase, MCK) 6,7. Grandes plasmides peuvent exprimer à des niveaux inférieurs. Le promoteur de CMV a été largement utilisé 6,7. </li><li> Purifier plasmides de E. coli en utilisant endotoxines conditions gratuits par les instructions du fabricant. Dissoudre le plasmide dans un tampon stérile, d&#39;endotoxine TE à 2 - 5 ug de plasmide / ul. </li><li> Diluer et aliquoter le plasmide à une Concentration de 20 ug d&#39;ADN dans 10 ul de TE endotoxine libre stérile (2 ug / ul) dans du tampon par injection d&#39;un tube de microcentrifugation stérile. Préparer une aliquote par injection. </li><li> Diluer le stock de hyaluronidase à 1x au phosphate solution saline stérile tamponnée sans calcium et le magnésium (PBS - / -) donnant une concentration finale de 8 unités. Aliquote de 10 pi de hyaluronidase dilué par injection dans un tube à centrifuger stérile. </li><li> Anesthésier la souris en utilisant 2,5% d&#39;isoflurane jusqu&#39;au sous sédation et ne répond pas à la queue ou de l&#39;orteil pincement. Placer l&#39;animal dans une position couchée sur le dos sur un coussin chauffant ou une table de chauffage pour maintenir la température normale du corps. </li><li> Nettoyer le coussinet plantaire de la souris avec une application alternée d&#39;un gommage povidone iode et de l&#39;alcool trois fois en utilisant une technique de préparation en provenance de l&#39;emplacement d&#39;aiguille et rayonnant vers l&#39;extérieur. Cette technique maintient le site de l&#39;aiguille à l&#39;état aseptique, minimisant introduction d&#39;agents pathogènes. </li><li> Injecter le coussinet plantaire de ee souris avec 10 ul de la solution de hyaluronidase 1x (2 mg / ml = 8 unités) entre la peau et le muscle en utilisant une seringue stérile de 1 ml. Hyaluronidase digère les composants de la matrice extracellulaire pour permettre l&#39;entrée plus efficace des myofibres dans le plasmide. Entrée à la base du talon et de faire avancer l&#39;aiguille vers les orteils. Relâchez lentement le liquide que l&#39;aiguille est rétracté. Le site d&#39;injection sous la peau va étendre et commencer à virer au rose. </li><li> Répétez les étapes 1.6 et 1.7 sur le pied controlatéral. </li><li> Débranchez l&#39;anesthésie et placez la souris de retour dans la cage. Laisser la souris pour se remettre complètement de l&#39;anesthésie. </li><li> Attendre 2 heures. </li><li> Répétez les procédures de stérilisation anesthésier et de pied, les étapes 1.5 et 1.6. </li><li> Injecter l&#39;ADN de plasmide dilué dans le coussinet plantaire de la même façon que la hyaluronidase. Assurez-vous que le volume maximum est de 20 pi ou moins. Pour l&#39;expression plasmidique double injecter 20 pg de chaque plasmide avec un volume maximum de 20 &amp;# 181; l ou moins. </li><li> Répéter l&#39;injection de plasmide sur la patte controlatérale ou utiliser le pied controlatéral pour les injections de contrôle. </li><li> Sur le pied qui a été initialement injecté, soulever la peau loin du muscle soulignant avec des pinces fines et placez une aiguille 27 G à travers les boules de peau près les orteils dans une position perpendiculaire à la ligne de guérir-orteil du pied. Prenez une seconde aiguille stérile 27 G et le placer horizontalement à travers la peau au niveau du talon. La place aiguilles parallèles entre elles ~ 1 cm. </li><li> En utilisant des pinces crocodiles, des électrodes de serrage aux aiguilles parallèles assurant que les aiguilles ne sont pas en contact les uns des autres. L&#39;utilisation de la pâte à modeler peut fixer les pinces en place. </li><li> Connectez électrodes à un stimulateur électrique. </li><li> Électroporer le muscle en appliquant 20 impulsions, 20 ms dans la durée / chacune, à 1 Hz, à 100 V / cm. Les orteils peuvent se contracter avec une stimulation. </li><li> Répétez la procédure de stimulation sur le pied controlatéral si nécessaire. </li><li>Retirer aiguilles. Essuyez le coussinet plantaire avec gommage povidone iodée. </li><li> Éteignez l&#39;anesthésie. Retour de la souris à la cage de récupération et de l&#39;activité de moniteur. Si la procédure a été menée comme prévu, l&#39;animal devrait reprendre la marche normale dans les 30 minutes et présentent pas de différence dans le niveau de la marche ou de l&#39;activité d&#39;un comportement pré-injection. Par conséquent, aucune analgésiques post-procédure sont nécessaires. Lors de la récupération, le réexpédier vers le vivarium. Note: L&#39;expression des protéines peut être testée dès l&#39;âge de 48 heures après l&#39;électroporation. Toutefois, pour permettre la récupération plus complète après l&#39;électroporation, nous préférons l&#39;isolement et l&#39;étude des myofibres 7 - 14 jours après l&#39;électroporation 10. L&#39;expression peut être dosée aussi longtemps que 4 semaines après l&#39;électroporation. </li></ol> 2. Procédure expérimentale pour l&#39;isolement du court fléchisseur des orteils (FDB) des fibres <ol><li> Collagénase dégel. Pour chaque faisceau FDB préparer deux puits dans une plaque de culture de tissu à 12 puits. Dans une bien ajouter 1 ml de préchauffé Eagles modifié milieu de Dulbecco, plus sérum-albumine bovine (DMEM + BSA) et dans l&#39;autre et ajouter 1 ml de solution de Ringer préchauffé. En outre, ajouter des frais 10 ml de solution de Ringer 1x à 35 mm plat Sylgard. </li><li> Sacrifier l&#39;animal par CO 2 ou un anesthésique par inhalation de gaz, suivie par dislocation cervicale ou une autre méthode pour garantir la mort. </li><li> Retirez les pieds postérieurs au-dessus de la cheville en utilisant une lame de rasoir propre et plonger dans une solution de Ringer 1x dans les 35 mm plat Sylgard. </li><li> Pin un pied serré, seule vers le haut, sur le plat Sylgard avec 30 g d&#39;aiguilles à travers chacune des 5 conseils d&#39;orteil et à la cheville. </li><li> À partir de la cheville, couper la peau vers le haut le côté du pied le long de la racine des cheveux vers les orteils, attention à ne pas entailler le muscle sous la peau. Répétez de l&#39;autre côté du pied. </li><li> Tenue de la peau à la base de la cheville avec une pince fine, couper la peau à travers le talon. </li><li> Lifting de la peaubattre avec des pinces, des ciseaux pointer vos vers la peau pour ne pas entailler le muscle. Snip le tissu conjonctif sous-jacent, éliminant efficacement la peau. </li><li> Pour retirer le faisceau musculaire FDB a coupé le gros tendon au talon pour libérer le tendon de l&#39;os. En tenant le tendon avec une pince, disséquer le faisceau FDB du gros tendon blanc enlevant soigneusement toute tissu conjonctif attaché à la FDB avec des ciseaux. Si fait correctement le faisceau facilement soulever des tendons sous-jacents révélant tendons blancs lumineux menant aux chiffres individuels. Couper le FDB les tendons près des orteils libérer le faisceau du pied. </li><li> Placer l&#39;ensemble du faisceau de muscle dans le puits contenant du milieu DMEM + BSA. </li><li> Répétez sur le pied controlatéral. </li><li> Une fois tous les muscles FDB sont isolés, ajouter 100 pi de la collagénase à chaque puits contenant DMEM + BSA et les muscles. </li><li> Vérifier le succès de l&#39;électroporation sur un microscope à fluorescence inversée avant la digestion. Si properl faity, vers le haut de 90% l&#39;efficacité de transfection peuvent être atteints. </li><li> Incuber la plaque à une humidifié, 37 ° C incubateur à 10% de CO 2 pendant environ 1 heure. Le temps d&#39;incubation peut varier selon le modèle de la maladie et le fond et le sort de la collagénase. </li><li> Après 1 h, transférer soigneusement le faisceau FDB à la solution ainsi contenant uniquement Ringers. </li><li> Triturer le faisceau FDB dans le puits avec la solution de Ringer en utilisant une pipette de 1 ml. Coupez l&#39;extrémité de la pointe de la pipette avec une lame de rasoir propre tels que le faisceau peut facilement passer à travers la pointe de la pipette. Triturer délicatement le muscle (~ 15 fois) permettant le faisceau de passer haut et en bas parallèlement à la pointe. Fibres intactes vont tomber dans la solution de Ringer. Lorsque les fibres ne tombent pas facilement le faisceau, placez le muscle du dos dans une solution de collagénase. </li><li> Plaque fibres isolées sur le plat ~ 500 pi par plaque. </li><li> Laisser le muscle à digérer dans le puits pendant 15 min. </li><li> Répétez les étapes 2.15de 2,17 jusqu&#39;à ce que le faisceau diminue en taille et la majorité des fibres sont dissociées à partir du faisceau (généralement un total de 2 à 3 fois). Une fois dissocié le muscle passe facilement à travers une pipette de 1 ml. </li><li> Laissez fibres fixer à la boîte pour un minimum de 15 min. Solution de Ringer frais peut être ajouté à la plaque pour diluer la collagénase résiduel ou modifiée en fonction de contraintes de temps. Remarque: Les fibres sont maintenant prêts pour l&#39;imagerie. </li></ol> 3. Procédure expérimentale pour la visualisation de la membrane induite par laser Dommages utilisant la microscopie confocale <ol><li> Fibres de charge immédiatement avant l&#39;imagerie avec 300 pi de 2,5 um FM 1-43 de FM 4-64 colorant dans une solution de Ringer. En général, les fibres d&#39;image 15 min à 2 h après l&#39;isolement. Cependant, les fibres peuvent être visualisés à l&#39;isolement après 24 h. </li><li> Placer le plat de fond de verre sur un microscope confocal équipé d&#39;un laser UV et un objectif 60X / 63X. </li><li> Localiser une fibre. Assurez-vous la fibre est fermement attachéau plat en tapant sur la base du microscope. Exclure les fibres qui ont été endommagés dans le processus de dissociation. Fibres endommagées peuvent contenir des vésicules membranaires, des niveaux élevés assimilation de colorant FM, boucle ou plier intensément. </li><li> Pour induire des lésions de la membrane sur la fibre, positionner le sarcolemme dans le centre de la fenêtre d&#39;imagerie avec un champ libre de noyaux. Focus sur le sarcolemme en utilisant le canal de colorant FM tels que le sarcolemme est extrêmement forte. </li><li> Placez les cheveux ROI de croix sur le sarcolemme utilisant le canal FM 4-64. </li><li> Irradier un point à 80% de puissance de 1 pixel pour 3 sec (~ 350 nm x 350 nm zone). Colorant FM sera entrer dans la cellule à l&#39;endroit de la blessure. Puissance et le temps peuvent être ajustés pour augmenter ou diminuer la zone de l&#39;insulte. </li><li> Capturez des images de la fibre avant que des dommages, au moment de dommages, tous les après-dommages 2 secondes, puis toutes les 10 secondes pour un maximum de 5 min. Timing peut être ajustée en fonction des besoins. Les images individuelles peuvent être convertis en films time-lapse dans J. Image </li><li> Répétez les étapes3.3 à 3.7 sur un maximum de 3 fibres par boîte. </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>

Il n&#39;y a pas de conflits d&#39;intérêts.

L&#39;utilisation d&#39;électroporation pour étudier les protéines marquées par fluorescence in vivo est idéalement adapté pour l&#39;étude du muscle étant donné l&#39;absence de modèles de lignée cellulaire appropriée qui reproduisent fidèlement la structure de la cellule musculaire entière. Ce protocole décrit l&#39;utilisation de l&#39;électroporation et la microscopie confocale à haute résolution pour fournir une image détaillée de la localisation de la protéine et la translocation apr?...

Myofibres individuels sont de grandes cellules syncytial, très organisée. Il ya quelques modèles de culture de cellules pour les muscles; Cependant, ces modèles ont comme limitation majeure qu&#39;ils ne différencient pas complètement dans les fibres musculaires matures. Par exemple, les lignées de cellules C2C12 et L6 sont dérivées de souris et les rats, respectivement 4.1. Dans des conditions de famine de sérum, les cellules mononucléaires &quot;de myoblastes comme« cesser la prolifération, subissent le retrait du cycle cellulaire, et entrent dans le programme myogénique former des cellules multinucléées avec sarcomères, appelées myotubes. Avec des conditions de culture prolongées, myotubes peuvent présenter des propriétés contractiles et &quot;contraction&quot; dans la culture. Lignées cellulaires humaines ont désormais été mis en place 5. En plus de ces lignées cellulaires immortalisées, myoblastes mononucléées peuvent être isolées de muscle et dans les conditions d&#39;inanition de sérum semblables formera myotubes. Ces lignées cellulaires et des cultures primaires de myoblastes sont hautement utiliserful car elles peuvent être transfectées avec des plasmides ou des virus et transduites utilisés pour étudier des processus biologiques cellulaires de base. Cependant, ces cellules, même lorsque induite pour former des myotubes, manquent beaucoup des traits saillants de l&#39;organisation musculaire mature. Plus précisément, les myotubes sont beaucoup plus petits que les fibres musculaires matures individuelles et manquent de la forme normale de fibres musculaires. Critique, myotubes manquent transversales (T-) tubules, le réseau membraneuse requis pour la libération de Ca efficace tout au long du myoplasme. Une autre méthode pour myoblastes primaires ou lignées de cellules myogéniques consiste à utiliser myofibres matures. La transgénèse peut être utilisé pour établir l&#39;expression de protéines marquées, mais cette méthode est coûteuse et prend du temps. In vivo électroporation de muscle de souris a émergé comme une méthode préférée pour sa vitesse et la fiabilité 6-10. Méthodes pour électroporation in vivo et l&#39;isolement efficace des myofibres haavez été optimisé pour le fléchisseur de la souris des orteils (FDB) 6 musculaire. Les procédés peuvent être effectués facilement et sont minimalement invasive pour induire l&#39;expression in vivo à partir de plasmides. Cette approche est maintenant combinée à des méthodes d&#39;imagerie à haute résolution, y compris l&#39;imagerie après rupture de laser de la 6,7 sarcolemme. La combinaison de colorants fluorescents et de l&#39;expression des protéines marquées par fluorescence de peut être utilisé pour surveiller des processus biologiques cellulaires en myofibres matures.

<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.

L&#39;électroporation est une technique peu invasive qui introduit efficacement ADN plasmidique purifié dans le court fléchisseur des orteils (FDB) faisceau musculaire (figure 1A). Sept jours après la transfection par fluorescence protéine marquée est visualisé dans les fibres musculaires isolées (figure 1B). Fibres isolées sont ensuite étalées et préparés pour l&#39;imagerie sur un microscope confocal. Les fibres peuvent être soumises à u...

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.

ADN électroporation, isolement et d&#39;imagerie de myofibres

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 vues.  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. 

Vidéo: DNA Electroporation, Isolation and Imaging of Myofibers

Isolation of Genomic DNA from Mouse Tails

Isolement de l&#39;ADN génomique de souris Tails

Recherche

Biologie

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.

Electroporation de mycobactéries

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.

Transfection d&#39;ADN des mammifères muscles squelettiques à l&#39;aide In Vivo L&#39;électroporation

 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.

Deux et trois dimensions imagerie de cellules vivantes de protéines endommagent l&#39;ADN de réponse

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.

Isolement et culture des fibres musculaires individuelles et de leurs cellules satellites du muscle squelettique adulte

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.

Analyse des embryonnaire et larvaire de poisson-zèbre squelettiques fibres musculaires de préparations dissociées

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.

La combinaison unique molécule manipulation et d&#39;imagerie pour l&#39;étude des interactions protéine-ADN

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.

Imagerie en temps réel de l&#39;unité d&#39;ingénierie des ARN transcrits dans des cellules vivantes utilisant Ratiométrique bimoléculaires 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.

Gravité réduite manifestations Environnement matérielles d&#39;un cytomètre de flux et Compagnon microfluidique technologie de mélange prototype miniaturisé

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.

Amplification, Séquençage de nouvelle génération, et l&#39;ADN génomique Cartographie des rétrovirales Sites d&#39;intégration

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