Name: dna electroporation, isolation and imaging of myofibers
Uploaded: 2015-12-23T15:00:00.000+00:00
Duration: 7 min 52 s
Description: Watch this Scientific Journal Video about DNA Electroporation, Isolation and Imaging of Myofibers at JoVE.com

Questo lavoro è stato sostenuto dal National Institutes of Health concede NS047726, NS072027, e AR052646.

I metodi in questo studio sono stati eseguiti in conformità con l&#39;etica Feinberg School of Medicine della Northwestern University Istituzionale Animal Care and Use Committee (IACUC) le linee guida approvate. Sono stati fatti tutti gli sforzi per ridurre al minimo le sofferenze. Procedura sperimentale 1. per elettroporazione in vivo del flessore Brevis (FDB) Muscle Bundle in mouse <ol><li> Plasmidi di progettazione per l&#39;espressione in vivo utilizzando un promotore saputo esprimere in cellule di mammifero (ad esempio, citomegalovirus, CMV) o nel muscolo (ad esempio, Muscolo Creatina chinasi, MCK) 6,7. Plasmidi più grandi possono esprimere a livelli inferiori. Il promotore CMV è stato ampiamente utilizzato 6,7. </li><li> Purificare plasmidi da E. coli utilizzando condizioni di libero endotossine le istruzioni del produttore. Sciogliere il plasmide in sterili, endotossine tampone TE libero a 2-5 mg plasmide / ml. </li><li> Diluire e un&#39;aliquota il plasmide di una concentration di 20 mg di DNA in 10 ml di endotossine sterile libero TE (2 mg / mL) tampone per iniezione in una provetta sterile. Preparare un&#39;aliquota per iniezione. </li><li> Diluire lo stock di ialuronidasi a 1x con sterile tampone fosfato senza calcio e magnesio (PBS - / -), dando una concentrazione finale di 8 unità. Aliquota 10 ml di ialuronidasi diluito per iniezione in una provetta sterile. </li><li> Anestetizzare il mouse utilizzando 2,5% isoflurano fino sedato e non risponde per la coda o pizzicare punta. Posto l&#39;animale in posizione supina su una piastra elettrica o una tabella di riscaldamento per mantenere la temperatura corporea normale. </li><li> Pulire la zampa del mouse con un programma alternato di uno scrub povidone iodio e alcol tre volte utilizzando una tecnica di preparazione proveniente dal sito di ago e irradia verso l&#39;esterno. Questa tecnica mantiene il sito ago in una condizione asettica, minimizzando introduzione patogeno. </li><li> Iniettare zampa di the mouse con 10 microlitri della soluzione 1x ialuronidasi (2 mg / ml = 8 unità) tra la pelle e il muscolo utilizzando una siringa sterile da 1 ml. Ialuronidasi digerisce componenti della matrice extracellulare per permettere l&#39;ingresso più efficiente del plasmide in miofibre. Invio alla base del tallone e far avanzare l&#39;ago verso le dita. Rilasciare lentamente il liquido mentre l&#39;ago viene retratto. Il sito di iniezione sotto la pelle si espanderà e inizia a diventare rosa. </li><li> Ripetere i punti 1.6 e 1.7 sul piede controlaterale. </li><li> Scollegare il anestesia e posizionare il mouse avanti nella gabbia. Lasciare che il mouse per riprendersi completamente dall&#39;anestesia. </li><li> Attendere 2 ore. </li><li> Ripetere le procedure di sterilizzazione e anestetizzante piede, punti 1.5 e 1.6. </li><li> Iniettare il DNA plasmidico diluita nella zampa nello stesso modo come la ialuronidasi. Assicurarsi che il volume massimo è di 20 ml o meno. Per l&#39;espressione dual plasmide iniettare 20 ug di ciascun plasmide con un volume massimo di 20 &amp;# 181; l o meno. </li><li> Ripetere l&#39;iniezione plasmide sulla zampa controlaterale o utilizzare il piede controlaterale per le iniezioni di controllo. </li><li> Sul piede che è stato inizialmente iniettato, sollevare la pelle dal muscolo sottolineare con pinza sottile e mettere un ago 27 G attraverso le sfere di pelle vicino le dita in posizione perpendicolare alla linea di guarire-punta del piede. Prendete un secondo sterili 27 ago G e posizionarlo in orizzontale attraverso la pelle sul tallone. Inserire aghi paralleli tra loro ~ 1 cm di distanza. </li><li> Utilizzando i morsetti a coccodrillo, elettrodi morsetto agli aghi paralleli assicurandosi gli aghi non siano a contatto tra loro. L&#39;uso della modellazione argilla può garantire i morsetti in posizione. </li><li> Collegare gli elettrodi di uno stimolatore elettrico. </li><li> Elettroporazione il muscolo applicando 20 impulsi, 20 msec di durata / ciascuno, a 1 Hz, 100 V / cm. Le dita possono contrarsi con la stimolazione. </li><li> Ripetere la procedura di stimolazione sul piede controlaterale, se necessario. </li><li>Rimuovere gli aghi. Pulire la zampa con iodio povidone macchia. </li><li> Spegnere l&#39;anestesia. Ritorna il mouse per la gabbia di recupero e monitorare l&#39;attività. Se la procedura è stata condotta come previsto, l&#39;animale deve riprendere il normale deambulazione entro 30 minuti e mostra alcuna differenza di deambulazione o un&#39;attività livello dal comportamento pre-iniezione. Pertanto, sono necessari analgesici post-procedura. Il recupero, rispedire l&#39;animale verso il vivaio. Nota: l&#39;espressione della proteina può essere saggiato fin da 48 ore dopo l&#39;elettroporazione. Tuttavia, per consentire il recupero più completo dopo l&#39;elettroporazione, noi preferiamo l&#39;isolamento e lo studio di miofibre 7 - 14 giorni dopo l&#39;elettroporazione 10. Espressione può essere saggiato fintanto 4 settimane dopo l&#39;elettroporazione. </li></ol> Procedura sperimentale per 2. Isolamento delle flessore Brevis (FDB) Fibre <ol><li> Collagenasi disgelo. Per ogni pacchetto FDB allestire due pozzetti in una piastra di coltura tissutale 12-bene. In un bene aggiungere 1 ml di pre-riscaldato Modified Eagles media Dulbecco più Bovine Serum Albumin (DMEM + BSA) e nell&#39;altra ben aggiungere 1 ml di pre-riscaldato soluzione Ringer. Inoltre, aggiungere freschi 10 ml di soluzione 1x Ringers ai 35 mm Sylgard piatto. </li><li> Sacrifica l&#39;animale attraverso CO 2 o inalazione di gas anestetico, seguita da dislocazione cervicale o un altro metodo per assicurare la morte. </li><li> Rimuovere le zampe posteriori sopra la caviglia con una lama di rasoio pulito e immergere in soluzione 1x Ringers nei 35 mm Sylgard piatto. </li><li> Pin un piede stretto, unico rivolto verso l&#39;alto, sul piatto Sylgard con 30 aghi da G a ciascuna delle punte 5 punta e alla caviglia. </li><li> Partendo alla caviglia, tagliare la pelle verso l&#39;alto il lato del piede lungo l&#39;attaccatura dei capelli verso le dita dei piedi, attenzione a non intaccare il muscolo sotto la pelle. Ripetere sull&#39;altro lato del piede. </li><li> Tenendo la pelle alla base della caviglia con una pinza sottile, tagliare la pelle attraverso il tallone. </li><li> Sollevamento la pellepatta con una pinza, puntare le forbici verso la pelle per non nick il muscolo. Tagliare il tessuto connettivo sottostante, rimuovere efficacemente la pelle. </li><li> Per rimuovere il fascio muscolare FDB tagliare il grande tendine del tallone per liberare il tendine dall&#39;osso. Tenendo il tendine con una pinza, sezionare il fascio FDB dal grande tendine bianco rimuovere accuratamente qualsiasi tessuto connettivo attaccato al FDB con le forbici. Se fatto correttamente il bundle sarà facile sollevare dei tendini sottostanti rivelano tendini bianchi brillanti che portano alle singole cifre. Tagliare la FDB ai tendini vicino le dita liberando il fascio dal piede. </li><li> Posizionare l&#39;intero fascio muscolare nel pozzo contenente DMEM + BSA. </li><li> Ripetere sul piede controlaterale. </li><li> Una volta che tutti i muscoli FDB sono isolate, aggiungere 100 ml di collagenasi in ciascun pozzetto contenente DMEM + BSA e muscoli. </li><li> Controllare il successo di elettroporazione con un microscopio a fluorescenza invertito prima della digestione. Se properl fattoy, verso l&#39;alto di 90% efficienza di trasfezione possono essere raggiunti. </li><li> Incubare la piastra in una umidificata, 37 ° C incubatore a 10% CO 2 per circa 1 ora. Il tempo di incubazione può variare a seconda del modello di malattia e lo sfondo e la sorte di collagenasi. </li><li> Dopo 1 ora, trasferire accuratamente il fascio FDB al pozzetto contenente unica soluzione di Ringer. </li><li> Triturare il fascio FDB nel pozzo con la soluzione Ringers con una pipetta 1 ml. Tagliare la fine della punta della pipetta con una lama di rasoio pulito in modo che il pacchetto può passare facilmente attraverso la punta della pipetta. Triturare delicatamente il muscolo (~ 15 volte) permettendo al fascio di passare su e giù parallelamente alla punta. Fibre intatte cadranno nella soluzione Ringers. Quando le fibre non facilmente cadere il fagotto, posizionare il muscolo posteriore in soluzione di collagenasi. </li><li> Piastra le fibre isolate sul piatto ~ 500 microlitri per piastra. </li><li> Lasciare il muscolo per digerire nel pozzo per 15 min. </li><li> Ripetere i punti 2.152.17 fino a fascio diminuisce dimensioni e la maggior parte delle fibre sono dissociato dal fascio (di solito un totale di 2-3 volte). Una volta dissociato il muscolo passa facilmente attraverso un puntale 1 ml. </li><li> Lasciate che le fibre si attaccano al piatto per un minimo di 15 min. Soluzione fresca Ringers può essere aggiunta alla piastra per diluire collagenasi residuo o cambiata a seconda vincoli di tempo. Nota: Le fibre sono ora pronti per l&#39;imaging. </li></ol> Procedura sperimentale 3. per la visualizzazione di laser-indotta danni della membrana utilizzando Microscopia confocale <ol><li> Fibre carico immediatamente prima di imaging con 300 ml di 2,5 micron FM 1-43 di FM 4-64 colorante in soluzione di Ringer. In generale, le fibre di immagine 15 minuti a 2 ore dopo l&#39;isolamento. Tuttavia, le fibre possono essere esposte fino a 24 ore dopo l&#39;isolamento. </li><li> Porre la capsula fondo di vetro su un microscopio confocale equipaggiato con un laser UV e un obiettivo 60X / 63X. </li><li> Individuare una fibra. Assicurarsi che la fibra è saldamente collegatoal piatto toccando la base del microscopio. Escludere le fibre che sono stati danneggiati nel processo di dissociazione. Le fibre danneggiate possono contenere blebs membrana, alti livelli di assorbimento di colorante FM, ricciolo o piegare intensamente. </li><li> Per indurre danni membrana sulla fibra, posizionare il sarcolemma al centro della finestra di esposizione con un campo libero di nuclei. Focus sul sarcolemma utilizzando il canale FM tintura in modo tale che il sarcolemma è estremamente tagliente. </li><li> Posizionare i capelli ROI croce sul sarcolemma utilizzando il canale FM 4-64. </li><li> Irradiare un punto di 1 pixel alla potenza di 80% per 3 secondi (~ 350 nm x 350 zona nm). Colorante FM entrerà nella cella nel sito di lesione. Potenza e ora possono essere modificate per aumentare o diminuire l&#39;area di insulto. </li><li> Cattura immagini della fibra prima danni, al momento del danno, ogni post-danni 2 sec, quindi ogni 10 sec per 5 min. Timing può essere regolata a seconda delle necessità. Le singole immagini possono essere convertite in filmati time-lapse in Image J. </li><li> Ripetere i passaggi3.3 attraverso 3.7 su un massimo di 3 fibre per piatto. </li></ol>

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L&#39;utilizzo di elettroporazione per studiare le proteine ​​fluorescenti tag in vivo è ideale per lo studio del muscolo in assenza di opportuni modelli di linee cellulari che riproducono fedelmente l&#39;intera struttura cellulare del muscolo. Questo protocollo descrive l&#39;utilizzo di elettroporazione e ad alta risoluzione microscopia confocale a fornire immagini dettagliate di localizzazione delle proteine ​​e la traslocazione dopo ferimento laser. Questo metodo può essere adattato per studiare ...

Miofibre individuali sono grandi, le cellule sinciziale altamente organizzati. Ci sono alcuni modelli di coltura cellulare per il muscolo; tuttavia, questi modelli hanno come principale limitazione che non fanno differenza completamente in fibre muscolari mature. Ad esempio, le linee cellulari C2C12 e L6 sono derivati ​​da topi e ratti, rispettivamente 1-4. In condizioni di deprivazione di siero, i mononucleari cellule &quot;mioblasti-like&quot; cessano proliferazione, subiscono il ritiro del ciclo cellulare, ed entrano nel programma miogenico che formano le cellule multinucleate con sarcomeri, indicati come miotubi. Con condizioni di coltura prolungata, miotubi possono esibire proprietà contrattili e &quot;contrazione&quot;, nella cultura. Linee cellulari umane hanno ormai stati istituiti 5. In aggiunta a queste linee cellulari immortalizzate, mioblasti mononucleate possono essere isolate dal muscolo e sotto le condizioni simili di deprivazione di siero formeranno miotubi. Queste linee cellulari e colture di mioblasti primari sono altamente usoFul perché possono essere trasfettate con plasmidi o trasdotte con virus e usate per studiare processi biologici cellulari di base. Tuttavia, queste cellule, anche se indotto a formare miotubi, mancano molte delle caratteristiche salienti di organizzazione muscolo maturo. In particolare, miotubi sono molto più piccoli singole fibre muscolari mature e non hanno la forma normale di miofibre. Criticamente, miotubi mancano trasversali (T-) tubuli, la rete membranosa richiesta per il rilascio del Ca 2+ efficiente in tutto il mioplasma. Un metodo alternativo per mioblasti primaria o linee cellulari miogeniche comporta usando fibre muscolari mature. Transgenesi può essere impiegato per stabilire espressione delle proteine ​​tag, ma questo metodo è costoso e richiede tempo. In vivo elettroporazione di muscoli del mouse è emerso come un metodo preferito per la sua velocità e affidabilità 6-10. Metodi per elettroporazione in vivo e l&#39;isolamento efficace di miofibre have stato ottimizzato per il mouse flessore brevis (FDB) muscolare 6. I metodi possono essere completate rapidamente e sono minimamente invasiva per indurre espressione vivo da plasmidi. Questo approccio è ora combinata con metodi di imaging ad alta risoluzione tra cui l&#39;imaging dopo interruzione del laser del 6,7 sarcolemma. La combinazione di coloranti fluorescenti e espressione di proteine ​​marcate fluorescenza può essere utilizzata per monitorare cellulari processi biologici miofibre mature.

<table><tbody><tr><td>DMEM</td><td>Life Technologies</td><td>11995-073</td><td>Dissection Media&lt;br/&gt; Dilution: 50 ml +100 g BSA</td></tr><tr><td>Collagenase, Type II</td><td>Life Technologies</td><td>17101-015</td><td>Fiber Digestion&lt;br/&gt; Dilution: 160 mg / 4 ml DMEM (stock 40 mg/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&lt;br/&gt; Dilution: 146 mM NaCl&lt;br/&gt; 5 mM&amp;nbsp;&amp;nbsp; KCl&lt;br/&gt; 2 mM&amp;nbsp;&amp;nbsp; CaCl2&lt;br/&gt; 1 mM&amp;nbsp;&amp;nbsp; MCl2&lt;br/&gt; 10 mM HEPES&lt;br/&gt; pH7.4 in H2O</td></tr><tr><td>1 ml Pipettes</td><td>Axygen</td><td>T-1000-C-R</td><td>Digestion</td></tr><tr><td>Razor Blades</td><td>Personna</td><td>94-120-71</td><td>Digestion</td></tr><tr><td>Precision Glide 30 G needle</td><td>BD Biosciences</td><td>305128</td><td>Dissection</td></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&lt;br/&gt; Dilution: 100 mg/50 ml DMEM</td></tr><tr><td>Falcon 60 mm dishes</td><td>Fisher Scientific</td><td>08772B</td><td>Dissection- used to hold sylgard</td></tr><tr><td>Clay</td><td></td><td></td><td>Electroporation</td></tr><tr><td>Stimulator&amp;nbsp;</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><td>Precision Glide 27 G needle</td><td>BD Biosciences</td><td>305136</td><td>Electroporation</td></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&lt;br/&gt; Dilution: Add 160 ul PBS (1x) to 40 &amp;micro;l 5x stock</td></tr><tr><td>1 cc U-100 Insulin Syringe (28G1/2)&amp;nbsp;</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>35 mm glass bottom Microwell dish</td><td>MaTek</td><td>P35G-1.5-14-C</td><td>Microscopy</td></tr><tr><td>FM 4-64</td><td>Life Technologies</td><td>T-13320</td><td>Microscopy&lt;br/&gt; Dilution: Final 2.5 ug/ml</td></tr><tr><td>FM 1-43</td><td>Life Technologies</td><td>T-35356</td><td>Microscopy&lt;br/&gt; 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></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.

Elettroporazione è una tecnica minimamente invasiva che introduce efficacemente purificato plasmide DNA nel flessore brevis (FDB) fascio muscolare (Figura 1A). Sette giorni dopo la transfezione fluorescently proteina tag viene visualizzato nella miofibre isolate (Figura 1B). Fibre isolate sono poi placcati e preparate per l&#39;imaging su un microscopio confocale. Le fibre possono essere sottoposti a lesione indotta da laser. Colorante FM può essere ut...

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.

Elettroporazione DNA, isolamento e imaging di miofibre

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 Views.  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. 

Video: DNA Electroporation, Isolation and Imaging of Myofibers

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Several mouse models have been created but they either do not truly represent the human physiopathology of the disease or are not representative of the broad spectrum of mutations found in humans. The immortalization of human primary myoblasts is an alternative to this limitation; however, it is still dependent on muscle biopsies, which are invasive and not easily available. In contrast, skin biopsies are easier to obtain and less invasive to patients. Fibroblasts derived from skin biopsies can be immortalized and transdifferentiated into myoblasts, providing a source of cells with excellent myogenic potential. Here, we describe a fast and direct reprogramming method of fibroblast into a myogenic lineage. Fibroblasts are transduced with two lentiviruses: hTERT to immortalize the primary culture and a tet-inducible MYOD, which upon the addition of doxycycline, induces the conversion of fibroblasts into myoblasts and then mature myotubes, which express late differentiation markers. This quick transdifferentiation protocol represents a powerful tool to investigate pathological mechanisms and to investigate innovative gene-based or pharmacological biotherapies for neuromuscular disorders.

This protocol describes the conversion of skin fibroblasts into myoblasts and their differentiation into myotubes. The cell lines are derived from patients with neuromuscular disorders and can be used to investigate pathological mechanisms and to test therapeutic strategies.

Riprogrammazione diretta dei fibroblasti umani in mioblasti per indagare le terapie per i disturbi neuromuscolari

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of ...

Ricerca

Genetica

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 DNA dei mammiferi muscoli scheletrici con In Vivo Elettroporazione

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

Biologia

Minimally Invasive Muscle Embedding (MIME) - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new muscle fibers under the right conditions. Although SCs can be harvested from muscle tissue and cultured in vitro, the resulting myoblast cells are not very effective in promoting myogenesis when transplanted into host muscle. Surgically exposing the host muscle and grafting segments of donor muscle tissue, or the isolated muscle fibers with their SCs onto host muscle, promotes better myogenesis compared to myoblast transplantation. We have developed a novel technique that we call Minimally Invasive Muscle Embedding (MIME). MIME involves passing a surgical needle through the host muscle, drawing a piece of donor muscle tissue through the needle track, and then leaving the donor tissue embedded in the host muscle so that it may act as a source of SCs for the host muscle. Here we describe in detail the steps involved in performing MIME in an immunodeficient mouse model that expresses a green fluorescent protein (GFP) in all of its cells. Immunodeficiency in the host mouse reduces the risk of immune rejection of the donor tissue, and GFP expression enables easy identification of the host muscle fibers (GFP+) and donor-cell-derived muscle fibers (GFP-). Our pilot data suggest that MIME can be used to implant an extensor digitorum longus (EDL) muscle from a donor mouse into the tibialis anterior (TA) muscle of a host mouse. Our data also suggest that when a myotoxin (barium chloride, BaCl2) is injected into the host muscle after MIME, there is evidence of donor-cell-derived myogenesis in the host muscle, with approximately 5%, 26%, 26% and 43% of the fibers in a single host TA muscle showing no host contribution, minimal host contribution, moderate host contribution, and maximal host contribution, respectively.

Minimally Invasive Muscle Embedding MIME - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

We describe a novel experimental technique that we call Minimally Invasive Muscle Embedding (MIME), which is based on the evidence that skeletal muscle tissue contains viable myogenic cells that can facilitate donor-cell-mediated myogenesis when implanted into a host muscle.

Minimamente invasiva muscolo incorporamento (MIME) - una nuova tecnica sperimentale per facilitare la miogenesi donatore-cellulo-mediata

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new ...

Medicina

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 la coltura di miofibre individuali e delle loro cellule satelliti da adulto muscolo scheletrico

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.

Analisi delle embrionali e larvali Zebrafish scheletriche miofibre da Preparazioni dissociata

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

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

Isolamento e cultura della miofibra singola da un modello Murine della distrofia muscolare di Emery-Dreifuss nei primi sviluppi post-Natal

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear positioning occurs during skeletal muscle differentiation. Muscle fibers (myofibers) are multinucleated cells formed by the fusion of muscle precursor cells (myoblasts) derived from muscle stem cells (satellite cells) that undergo proliferation and differentiation. Correct nuclear positioning within myofibers is required for the proper muscle regeneration and function. The common procedure to assess myoblast differentiation and myofiber formation relies on fixed cells analyzed by immunofluorescence, which impedes the study of nuclear movement and cell behavior over time. Here, we describe a method for the analysis of myoblast differentiation and myofiber formation by live cell imaging. We provide a software for automated nuclear tracking to obtain a high-throughput quantitative characterization of nuclear dynamics and myoblast behavior (i.e., the trajectory) during differentiation and fusion.

Skeletal muscle differentiation is a highly dynamic process, which particularly relies on nuclear positioning. Here, we describe a method to track nuclei movements by live cell imaging during myoblast differentiation and myotube formation and to perform a quantitative characterization of nuclei dynamics by extracting information from automatic tracking.

Sfruttando Live Imaging ai Nuclei di pista durante il differenziamento dei mioblasti e Fusion

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear ...

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

Isolamento di Myofibrils da biopsie muscolari scheletriche e determinazione della funzione Contractile con un trasduttore della forza di risoluzione Nano-Newton

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

Test di coltura di una singola miofibra per la valutazione della funzionalità delle cellule staminali muscolari adulte Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

Isolamento enzimatico e caratterizzazione di fibre muscolari di topo a lunghezza variabile

Intact Short, Intermediate, and Long Skeletal Muscle Fibers Obtained by Enzymatic Dissociation of Six Hindlimb Muscles of Mice: Beyond Flexor Digitorum Brevis

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal with the short fibers of the flexor digitorum brevis (FDB), which restrains the scope of results dealing with fiber types, limits the amount of biological material available, and impedes a clear connection between cellular physiological phenomena and previous biochemical and dynamical knowledge obtained in other muscles.
This paper describes how to obtain intact fibers from six muscles with different fiber type profiles and lengths. Using C57BL/6 adult mice, we show the muscle dissection and fiber isolation protocol and demonstrate the suitability of the fibers for Ca2+ transient studies and their morphometric characterization. The fiber type composition of the muscles is also presented. When dissociated, all muscles rendered intact, living fibers that contract briskly for more than 24 h. FDB gave short (&#60;1 mm), peroneus digiti quarti (PDQA) and peroneus longus (PL) gave intermediate (1-3 mm), while extensor digitorum longus (EDL), extensor hallucis longus (EHL), and soleus muscles released long (3-6 mm) fibers.
When recorded with the fast dye Mag-Fluo-4, Ca2+ transients of PDQA, PL, and EHL fibers showed the fast, narrow kinetics reminiscent of the morphology type II (MT-II), known to correspond to type IIX and IIB fibers. This is consistent with the fact that these muscles have over 90% of type II fibers compared with FDB (~80%) and soleus (~65%). Moving beyond FDB, we demonstrate for the first time the dissociation of several muscles, which render fibers spanning a range of lengths between 1 and 6 mm. These fibers are viable and give fast Ca2+ transients, indicating that the MT-II can be generalized to IIX and IIB fast fibers, regardless of their muscle source. These results increase the availability of models for mature skeletal muscle studies.

Autore in primo piano: Isolamento delle fibre muscolari lunghe dai muscoli degli arti posteriori di topo per lo studio dell'accoppiamento eccitazione-contrazione tra i tipi di fibre

We describe a protocol to obtain enzymatically dissociated fibers of different lengths and types from six muscles of adult mice: three of them already described (flexor digitorum brevis, extensor digitorum longus, soleus) and three of them successfully dissociated for the first time (extensor hallucis longus, peroneus longus, peroneus digiti quarti).

Fibre muscolari scheletriche corte, intermedie e lunghe intatte ottenute dalla dissociazione enzimatica di sei muscoli degli arti posteriori di topi: oltre il flessore breve delle dita

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal ...