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+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.

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

Isolation of Genomic DNA from Mouse Tails

Fare Tails Genomik DNA izolasyonu

Biyoloji

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.

Mikobakteriler, Elektroporasyon

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.

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 ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

Kraniofasial mezenşimin, Elektroporasyon

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

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.

DNA Hasarı Tepki Proteinler İki ve Üç Boyutlu Canlı Hücre Görüntüleme

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.

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 ...

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.

Protein-DNA Etkileşiminin Çalışmaları Tek molekül Manipülasyon ve Görüntüleme birleştiren

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.

Oranlı metrik Bimoleküler Fenerleri kullanma Yaşam Hücrelerde Tek Engineered RNA Transkriptlerinin Gerçek zamanlı Görüntüleme

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.

Prototip Minyatür Akış Sitometresi ve Companion mikroakışkan Karıştırma Teknoloji İndirimli-gravite Çevre Donanım Gösterileri

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