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

Bu çalışma NS047726, NS072027 ve AR052646 Ulusal Sağlık Enstitüleri tarafından hibe desteklenmiştir.

Bu çalışmada yöntemleri kuralları onayladı Tıp Kurumsal Hayvan Bakımı ve Kullanımı Komitesi Northwestern Üniversitesi Feinberg School (IACUC) etik uygun olarak yapıldı. Tüm çabalar acıyı en aza indirmek için yapılmıştır. Fare Flexor Digitorum Brevis in vivo Elektroporasyon (FDB) kas Bundle 1. Deney Prosedürü <ol><li> Memeli hücrelerinde eksprese ettiği bilinen bir promoteri kullanılarak in vivo ifadesi için plazmidler tasarım (örneğin, sitomegalovirüs, CMV) ya da kas (örneğin, kas kreatin kinaz, MCK) 6,7. Büyük plazmidler daha düşük seviyelerde ifade edebilir. CMV promoteri yoğun 6,7 kullanılmıştır. </li><li> E. plazmidleri arıtın E. coli, üretici talimatlarına göre endotoksin serbest koşulları kullanılarak hazırlanabilir. 5 ug plazmid / ul - 2 steril, endotoksin serbest TE tamponu içinde plazmid çözülür. </li><li> Seyreltik bir concentratio plasmiti bölmeyinSteril endotoksin serbest TE (2 ug / ml) 10 ul DNA 20 ug n steril mikrosantrifüj tüpü içine enjeksiyon başına tampon. Enjeksiyon başına bir kısım hazırlayın. </li><li> 8 adet nihai yoğunluk, kalsiyum ve magnezyum olmaksızın, steril fosfat tamponlu salin ile 1x hyaluronidaz stok - (- / PBS) ile seyreltilir. Kısım steril bir mikrosantrifüj tüpe enjeksiyon başına seyreltilmiş hiyalüronidaz 10 ul. </li><li> Sedasyon ve kuyruk veya ayak sıkışmasını yanıt vermeyen kadar% 2.5 izofluran kullanarak fare anestezisi. Normal vücut sıcaklığının muhafaza edilmesi için bir ısıtma yastığı veya ısıtma masasına sırt üstü yatırıldı pozisyonda hayvan yerleştirin. </li><li> Bir hazırlık teknik, iğne buradan kaynaklanan ve dışarı doğru yayılan kullanarak bir povidon iyodin fırçalama ve alkol bir alternatif uygulaması ile üç kez fare ayak desteği temizleyin. Bu teknik, patojen giriş en aza indirerek, bir aseptik durumda iğne sitesine sahiptir. </li><li> Th Kapkaççı enjektederi ve steril bir 1 ml şırınga ile kas arasındaki 1x hiyaluronidaz çözeltisi (2 mg / ml = 8 birim) 10 ul e fare. Hyaluronidaz myofibers içine plasmidin daha verimli bir giriş izin vermek için hücre dışı matris bileşenleri sindirir. Topuk üssünde girin ve ayak parmakları doğru iğne ilerlemek. Iğne çekilir yavaş yavaş sıvı bırakın. Deri altına enjeksiyon sitesi genişletmek ve pembe açmak başlayacaktır. </li><li> Tekrarlayın 1.6 ve kontralateral ayak üzerinde 1.7 adımları yineleyin. </li><li> Anestezi ayırın ve tekrar kafese fareyi koyun. Fare tam anestezi kurtarmak için izin verin. </li><li> 2 saat bekleyin. </li><li> , Anestezi ve ayak sterilizasyon prosedürleri tekrarlayın 1.5 ve 1.6 adımları tekrarlayın. </li><li> Hyaluronidase ile aynı şekilde ayak tabanı içine seyreltildi plazmid DNA enjekte edilir. Maksimum hacmi 20 ul veya daha az olduğundan emin olun. Çift plazmid ekspresyon için 20 ve en fazla hacimde her plazmid 20 ug enjekte# 181; l veya daha az. </li><li> Kontralateral ayak tabanı üzerinde plazmid enjeksiyonu tekrarlayın veya kontrol enjeksiyonlar için kontralateral ayak kullanın. </li><li> Başlangıçta enjekte edildi yürüyerek uzak ince forseps ile altını kas cildi kaldırın ve ayak iyileşmek-ayak hattına dik bir pozisyonda ayak yakınında cilt topları ile biri 27 G iğne yerleştirin. İkinci steril 27 G iğne alın ve topuk deri yoluyla yatay olarak yerleştirin. Yer iğneler dışında her ~ diğer 1 cm paralel. </li><li> Iğneler birbirine temas etmeyecek emin paralel iğnelere timsah kelepçeleri, kelepçe elektrotlar kullanarak. Kil modelleme kullanımı yerine kelepçeler güvence altına alabilirsiniz. </li><li> Bir elektrik stimülatörü elektrotları bağlayın. </li><li> 100 V / cm, 1 Hz, 20 baklagiller, süresi 20 milisaniye / her uygulayarak kas Electroporate. Ayak stimülasyonu ile seğirme olabilir. </li><li> Gerekirse kontralateral ayak stimülasyon işlemi tekrarlayın. </li><li>Iğneler çıkarın. Povidon iyot ile ovulur Kapkaççı silin. </li><li> Anestezi kapatın. Kurtarma kafes ve monitör aktivite fare dönün. Beklendiği gibi işlem yapılmıştır ise, hayvan 30 dakika içinde normal ambulasyon kazanması ve enjeksiyon öncesi davranışından ambulasyon veya aktivite düzeyi arasında fark göstermelidir. Bu nedenle, hiçbir işlem sonrası analjezikler ihtiyaç vardır. Kurtarma üzerine, vivaryum hayvan dönün. Not: Protein ifadesi, elektroporasyon sonra kadar erken olarak 48 saat analiz edilebilir. 14 gün elektroporasyon 10 sonra - Bununla birlikte, elektroporasyon sonra daha tam iyileşme sağlamak için, biz 7 tecrit ve myofibers çalışma tercih ederim. İfade 4 hafta sonra, elektroporasyon sürece analiz edilebilir. </li></ol> Fleksör Digitorum Brevis (FDB) Elyaf İzolasyonu 2. Deney Prosedürü <ol><li> Çözülme kollajenaz. Her bir paket için FDB 12 oyuklu doku kültürü plakası, iki kuyu hazırlar. Birinde de 1 m eklemekÖnceden ısıtılmış Dulbecco Değiştirilmiş Kartal Ortamı artı sığır serum albümini (DMEM + BSA) ve l de önceden ısıtılmış Ringers Çözeltisinin 1 ml. Ayrıca, çanak Sylgard 35 mm 1x Ringer solüsyonu taze 10 ml ekleyin. </li><li> Ölümü sağlamak için servikal dislokasyon veya başka bir yöntemle takip CO 2 veya anestezik gaz soluma yoluyla hayvan kurban. </li><li> Temiz bir jilet kullanarak ayak bileği eklemi üzerinde arka ayakları çıkarın ve çanak Sylgard 35 mm 1x Zil çözeltide daldırın. </li><li> Tek ayak sıkı Pin, 5 parmak uçları her yoluyla ve ayak bileğinde 30 G iğne ile Sylgard çanak üzerine, tek yukarı bakacak. </li><li> Ayak bileğinde başlayan, dikkatli nick, düz ayak doğru saç çizgisi boyunca ayak tarafı yukarı derinin altında kas cilt snip. Ayağın diğer tarafta tekrarlayın. </li><li> Ince forseps ile ayak bileği üssünde cilt tutan, topuk boyunca cildi kesti. </li><li> Cildi KaldırmaCilde olarak değil nick kas doğru makas gelin forseps ile flep. Cildi etkili kaldırarak, altta yatan bağ dokusu snip. </li><li> FDB kas demeti kemikten tendon ücretsiz topuk büyük tendonu kesilmiş kaldırın. Forseps ile tendonu tutarak dikkatle makasla FDB bağlı herhangi bir bağ dokusu kaldırarak büyük beyaz tendon gelen FDB paket teşrih. Düzgün yapılırsa paket kolayca bireysel basamak önde gelen parlak beyaz tendonları ortaya yatan tendonların kapalı kaldıracak. Ayak paket kurtararak ayak yakınında tendon FDB kesin. </li><li> Iyi içeren DMEM + BSA bütün kas demeti koyun. </li><li> Kontralateral ayak üzerinde tekrarlayın. </li><li> Bir kez tüm FDB kasları her kuyuya içeren DMEM + BSA ve kas kollajenaz 100 ul ekleyin izole edilmiştir. </li><li> Sindirim öncesinde bir ters flüoresan mikroskop elektroporasyon başarısını kontrol edin. Yapılan properl Eğery,% 90 transfeksiyon randımanının yukarı doğru elde edilebilir. </li><li> Yaklaşık olarak 1 saat süre ile,% 10 CO2 nemlendirilmiş bir 37 ° C kuluçka makinesi içinde plaka inkübe edin. Kuluçka süresi hastalık modeli ve arka plan ve kolajenaz partisine bağlı olarak değişebilir. </li><li> 1 saat sonra, dikkatli bir şekilde de ihtiva eden tek Ringers çözeltisi FDB paket aktarın. </li><li> 1 ml pipet kullanarak Ringer çözeltisi ile iyi FDB paket Karışım. Paket kolaylıkla pipet geçebilir şekilde temiz bir jilet ile pipet ucu kesilir. Yavaşça paket kaçırmak ve bahşiş paralel aşağı izin kas (~ 15 kez) çiğnemek. Bozulmamış lifler Ringer Çözüm içine kapalı düşecek. Lifler kolayca paket dökmez zaman kollajenaz çözüm geri kas yerleştirin. </li><li> Plaka başına çanak ~ 500 ul izole lifleri Plate. </li><li> Kas 15 dakika boyunca oyuk sindirimi izin verin. </li><li> Yineleyin 2.15 adımları2.17 kadar paket boyutu düşene kadar ve liflerin çoğunluğu demetinin (2-3 kat genellikle toplam) ayrışmış edilir. Bir kez kas kolayca 1 ml pipet ucu geçer ayrışmış. </li><li> Lifler 15 dakika en az çanak eklemek olsun. Taze Ringers çözüm zaman kısıtlamaları bağlı kalan kollajenaz sulandırmak için plakaya eklenebilir veya değiştirilebilir. Not: fiberler şimdi görüntüleme için hazırız. </li></ol> Konfokal Mikroskobu kullanılarak lazer kaynaklı Membran Hasar Görselleştirme 3. Deneysel Prosedürü <ol><li> Yük lifler 2,5 uM, 300 ul Ringer çözeltisi içinde FM 4-64 boya FM 1-43 ile görüntüleme hemen önce. Genellikle, izolasyondan sonra görüntü lifleri 15 dakika 2 saat. Ancak, lifler 24 saat sonrası izolasyon kadar görüntülü olabilir. </li><li> UV lazer ve 60X / 63X objektif ile donatılmış bir konfokal mikroskop cam alt çanak yerleştirin. </li><li> Bir fiber bulun. Lif sıkıca tutturulmuş olduğundan emin olunmikroskop tabanını dokunarak çanak. Ayrışma sürecinde zarar görmüş lifler hariç. Hasarlı lifler kıvırmak zar kabarcıklar, FM boya alımı yüksek düzeyde içeren veya yoğun bükülebilir. </li><li> Fiber membran hasarına neden için, çekirdeklerin net bir alana sahip görüntüleme penceresinin ortasında sarcolemma yerleştirin. Sarcolemma son derece keskin şekilde FM boya kanalını kullanarak sarcolemma odaklanın. </li><li> FM 4-64 kanalı kullanan sarcolemma ROI çapraz saç yerleştirin. </li><li> 3 sn (~ 350 nm alanı x 350 nm) için% 80 güçte bir 1 piksel noktası ışın tedavisi. FM boya yaralanma yerinde hücreye girer. Güç ve zaman artırmak veya hakaret alanını azaltmak için ayarlanabilir. </li><li> , Hasar anındaki hasar öncesi her 2 sn sonrası hasar, en fazla 5 dakika süreyle daha sonra her 10 saniyede bir lif görüntüler yakalayın. Zamanlama ihtiyaca göre ayarlanabilir. Bireysel görüntüleri Görüntü J. zaman atlamalı filmleri dönüştürülebilir </li><li> Adımları tekrarlayınTabak başına en fazla 3 lifleri üzerinde 3.7 ile 3.3. </li></ol>

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Herhangi bir çıkar çatışması vardır.

In vivo olarak, floresan etiketli proteinler çalışma elektroporasyon kullanımı ideal sadakatle kas tüm hücre yapısını taklit Uygun bir hüre hattı modellerinin olmaması ile kas çalışma için uygundur. Bu protokol lazer yaralama sonra protein lokalizasyonu ve translokasyon ayrıntılı görüntüleme sağlamak için elektroporasyon ve yüksek çözünürlüklü konfokal mikroskopi kullanımını açıklar. Bu yöntem hücre iskeleti yeniden, insan ticareti ve füzyon gibi diğer hücresel süreçl...

Bireysel myofibers büyük, çok organize sinsityal hücrelerdir. Kas birkaç hücre kültürü modeli vardır; Ancak, bu modeller tam olgun myofibers içine ayırt yok onların ana sınırlama olarak var. Örneğin, C2C12 ve L6 hücre çizgileri farelerde ve sıçanlarda, sırası ile 1-4 elde edilir. Serum açlık koşullarında, tek çekirdekli &quot;miyoblast gibi&quot; hücreler çoğalması ateşkes hücre döngüsü çekilmesi geçmesi ve sarkomer ile çok çekirdekli hücreler oluşturan Miyojenik programa girmek, miyotüplerinin olarak anılacaktır. Uzun süreli kültür koşulları ile miyotüpleri kasılma özellikler gösterebilir ve kültürde &quot;seğirme&quot;. İnsan hücre hatları da hemen 5 tespit edilmiştir. Bu ölümsüzleştirilmiş hücre hatlarına ek olarak, tek çekirdekli miyoblastlar kas ve miyotüpler oluşturacak serum açlık benzer koşullar altında izole edilebilir. Bu hücre hatları ve birincil miyoblast kültürleri çok kullanımı vardırful bu plasmidler ile transfekte ya da virüs ile transduse ve temel hücre biyolojik süreçleri incelemek için kullanılabilir, çünkü. Miyotüpler oluşturmak için uyarılan Ancak, bu hücreler, hatta, olgun kas örgütün belirgin özelliklerinden birçoğu yoksundur. Özellikle, miyotüpleri bireysel olgun myofibers çok daha küçüktür ve myofibers normal şeklini yoksundur. Kritik, miyotüpleri enine (T) tübüller eksikliği, myoplasm boyunca verimli Ca 2+ serbest bırakılması için gerekli membranöz ağ. Birincil miyoblast veya myojenik hücre hatlarına alternatif bir yöntem olgun myofibers kullanarak gerektirir. Transgenesis etiketli proteinlerin ekspresyonunu belirlemek için kullanılabilir, ancak bu yöntem pahalı ve zaman alıcı bir iştir. Fare kas in vivo elektroporasyon hızı ve güvenilirliği 6-10 için tercih edilen bir yöntem olarak ortaya çıkmıştır. In vivo elektroporasyon ve myofibers ha etkin izolasyon yöntemleriFare fleksör digitorum brevis (FDB) kas 6 için optimize oldum. Yöntemler hali hazırda tamamlanmış ve plazmidlerden in vivo ifadenin uyarılması için minimal invaziv girmektedir. Bu yaklaşım artık sarcolemma 6,7 lazer bozulması sonrasında görüntüleme dahil olmak üzere yüksek çözünürlüklü görüntüleme yöntemleri ile kombine edilir. Floresan boyalar ve floresan etiketli proteinlerin ekspresyonu kombinasyonu, olgun myofibers hücre, biyolojik işlemleri izlemek için de kullanılabilir.

<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.

Elektroporasyon verimli fleksör digitorum brevis (FDB) kas demeti (Şekil 1A) içine saflaştırılmış plazmid DNA tanıtır minimal invaziv bir tekniktir. Yedi gün transfeksiyon floresan etiketli protein izole myofibers (Şekil 1B) görüntülenmiştir yazı. İzole edilmiş lifler, daha sonra kaplama ve konfokal mikroskop görüntüleme için hazırlanır. Fiberler, lazer kaynaklı yaralanmalara maruz bırakılabilir. FM boya membran zararının al...

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

DNA Electroporation, Isolation and Imaging of Myofibers

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

DNA Elektroporasyon, Myofibers İzolasyon ve Görüntüleme

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 Görüntüleme.  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.

Nöromüsküler Bozukluklar için Tedavileri Araştırmak için İnsan Fibroblastlarının Myoblastlara Doğrudan Yeniden Programlanması

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

Genetik

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.

Memeli İskelet Kaslar kullanarak DNA Transfeksiyon In Vivo Elektroporasyon

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

Biyoloji

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.

Minimal invaziv kas (MIME) katıştırma - donör-Hücre-aracılı Myogenesis kolaylaştırmak için yeni bir deneysel teknik

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

Tıp

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.

Yetişkin İskelet Kası gelen bireysel Myofibers ve Uydu Hücre İzolasyonu ve Kültür

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.

Disosiye Müstahzarlar gelen Embriyonik ve larval Zebra balığı İskelet myofibers Analizi

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).

Erken Doğum Sonrası Gelişimde Emery-Dreifuss Musküler Distrofi'nin Murine Modelinden Tek Miyofiber İzolasyon ve Kültür

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.

Parça çekirdeği canlı görüntüleme sırasında Myoblast farklılaşma ve füzyon istismar

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.

İskelet Kas Biyopsilerinden Miyofibrillerin İzlenmesi ve Nano-Newton Çözünürlük Transdüseri ile Kontraktil Fonksiyonun Belirlenmesi

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.

Yetişkin Kas Kök Hücre İşlevselliğinin Değerlendirilmesi için Tek Miyofiber Kültür Tahlil 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 ...

Değişken Uzunluklu Fare Kas Liflerinin Enzimatik İzolasyonu ve Karakterizasyonu

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.

Yazar Spot Işığı: Lif Tipleri Arasında Uyarma-Kasılma Kuplajını İncelemek için Fare Arka Bacak Kaslarından Uzun Kas Liflerinin İzolasyonu

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).

Farelerin Altı Arka Bacak Kasının Enzimatik Ayrışmasıyla Elde Edilen Sağlam Kısa, Orta ve Uzun İskelet Kası Lifleri: Fleksör Digitorum Brevis'in Ötesinde

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