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

这项工作是支持的美国国立卫生研究院授予NS047726，NS072027和AR052646。

本研究的方法是在道德根据医学实验动物管理和使用委员会的西北大学Feinberg医学院（IACUC）批准执行准则。所作的所有努力，以尽量减少痛苦。 对于趾短屈肌的体内电 （FDB）肌束在小鼠1.实验步骤<ol><li>使用已知的在哺乳动物细胞中表达的启动子设计的质粒的体内表达 （例如，巨细胞病毒，巨细胞病毒），或在肌肉（例如，肌肉肌酸激酶，MCK）6,7。较大的质粒可以表达在较低的水平。在CMV启动子已经被广泛地使用6,7。 </li><li>净化从大肠杆菌的质粒使用大肠杆菌每制造商的说明无内毒素的条件。溶解在无菌，无内毒素TE缓冲质粒在2 - 5微克质粒/μL。 </li><li>稀释和分装的质粒到concentratio于10μl无菌无内毒素的TE（2微克/微升）正20微克的DNA每次注射缓冲到无菌离心管中。准备每次注射一个等份。 </li><li>稀透明质酸酶的库存至1x用无菌磷酸盐缓冲盐水不含钙和镁（PBS - / - ），得到的8个单位的终浓度。等份加入10μl每次注射稀释透明质酸到无菌离心管中。 </li><li>麻醉用2.5％异氟醚，直到镇静和反应迟钝尾巴或脚趾捏鼠标。将动物在加热板上或加热表仰卧位置，以保持正常体温。 </li><li>使用制备型技术从针站点始发和向外辐射清洁鼠标的足垫用聚维酮碘擦洗和醇的交替应用三次。这一技术保持针站点在无菌条件下，最大限度地减少病原体的介绍。 </li><li>注入日的足垫E滑鼠与10微升的皮肤，并用无菌的1ml注射器的肌肉之间的1倍的透明质酸酶溶液（2毫克/毫升= 8单位）。透明质酸酶消化细胞外基质的成分，以允许更有效的进入质粒的成肌纤维。输入在脚后跟的基部和推进针朝向脚趾。缓慢释放的液体当针缩回。皮肤下的注射部位将扩大，并开始变成粉红色。 </li><li>重复步骤1.6和1.7对侧脚。 </li><li>断开麻醉，然后将鼠标放回笼子。让鼠标完全从麻醉中恢复。 </li><li>等待2小时。 </li><li>重复麻醉和脚消毒程序，步骤1.5和1.6。 </li><li>注入稀释质粒DNA导入以相同的方式如透明质酸酶的足垫。确保最大音量为20微升或更少。对于双质粒表达注资20微克每个质粒与20的最大容积＃181; l或更少。 </li><li>重复对侧足垫质粒注射或使用对侧脚控制注射。 </li><li>上，最初注入的脚，抬起皮肤远离强调肌肉用细镊子和放置一个27G的针头通过邻近脚趾的皮肤中的球的位置垂直于脚的愈合趾线。花一秒钟无菌27G的针头和水平放置它通过皮肤的脚跟。地方针彼此平行〜1厘米分开。 </li><li>用鳄鱼钳，钳电极平行针，确保针不接触对方。使用橡皮泥可以保护夹到位。 </li><li>连接到电极的电刺激器。 </li><li>通过施加20个脉冲，20毫秒持续时间/每个，在1Hz，在100伏/厘米电穿孔肌肉。脚趾可能会抽搐与刺激。 </li><li>如果需要的话，重复的刺激方法对侧脚。 </li><li>取出针。请用碘伏擦洗脚垫。 </li><li>关闭麻醉。返回鼠标到恢复笼和监控活动。如果该程序是进行如预期的动物应恢复正常下地在30分钟内，并表现出从预喷射行为在行走或活性水平没有差异。因此，需要没有后置过程镇痛药。恢复后，返回动物到动物饲养。 注:蛋白质表达可以电穿孔后进行测定早48小时。然而，让电后更完全恢复，我们更倾向于孤立和肌纤维的研究7 -电10日之后 14天。表达可用来测定，只要电穿孔后4周。 </li></ol>对于趾短屈肌（FDB）纤维分离2.实验过程<ol><li>解冻胶原酶。对于每一个FDB捆在一个12孔培养板准备两口井。在一个孔加1米升预热的Dulbecco氏改良的Eagles介质加牛血清白蛋白（DMEM + BSA）和在其它公添加预热的林格氏溶液1毫升。此外，加入新的10毫升1X林格氏液，以35毫米的Sylgard菜。 </li><li>通过CO 2或麻醉气体吸入牺牲动物，颈椎脱位或其它方法，以确保死亡。 </li><li>使用干净的刀片卸下后英尺以上的踝关节和淹没在35毫米培养皿SYLGARD 1×林格溶液。 </li><li>脚一只脚紧，鞋底朝上，上的Sylgard菜通过各5足尖和脚踝地下30针。 </li><li>开始在脚踝，剪断皮肤向上伸直脚沿着朝向脚趾发际线的一侧，注意不要切口皮下的肌肉。重复上脚的另一侧。 </li><li>保持皮肤在含有细镊子脚踝的基础上，跨越足跟皮肤。 </li><li>提升肌肤瓣钳，指向你的剪刀朝皮肤不尼克肌肉。剪断底层结缔组织，有效去除皮肤。 </li><li>要删除FDB肌束切大牛筋鞋跟释放从骨肌腱。保持用钳子肌腱，解剖FDB束从白色大腱小心除去附着在FDB用剪刀任何结缔组织。如果处理得当的包很容易取下底层筋透出明亮的白色筋导致个别数字的。切FDB在靠近脚趾释放从脚被捆绑的肌腱。 </li><li>发生在井的DMEM + BSA整个肌束。 </li><li>重复对侧脚。 </li><li>一旦所有FDB肌肉分离，添加100微升胶原酶到每孔含有DMEM + BSA和肌肉。 </li><li>检查电对之前消化一个倒置荧光显微镜成功。如果做得properlY，90％的转染效率向上可以实现。 </li><li>孵育板在湿润的37℃培养箱中，在10％CO 2的约1小时。根据疾病模型和背景和很多胶原酶的孵育时间可以变化。 </li><li> 1小时后，小心地将FDB束传送到井仅含林格溶液。 </li><li>磨碎FDB束在井中使用1毫升吸移管的林格溶液。切枪头用干净的刀片，使得包可以通过移液管尖端容易地通过的末端。轻轻磨碎的肌肉（〜15倍），允许包通过上下平行提示。完整的纤维会脱落进林格氏液。当纤维不容易脱落的捆绑，把肌肉放回胶原酶溶液。 </li><li>板隔离纤维上的菜〜500微升每盘。 </li><li>允许肌肉在井消化15分钟。 </li><li>重复步骤2.15到2.17，直到束在尺寸减小，并且大部分的纤维从束（通常是一个总的2-3倍）解离。一旦分离肌肉容易通过1毫升的枪头。 </li><li>让纤维附着到培养皿为至少15分钟。新鲜林格氏溶液可以被添加到该板以稀释残余的胶原酶或改变取决于时间的限制。 注:纤维是现在准备成像。 </li></ol>利用共聚焦显微镜的激光诱导膜损伤可视化3.实验步骤<ol><li>负载纤维成像300微升2.5微米的FM 4-64染料林格氏液FM 1-43前夕。通常，分离后的图像的纤维15分钟到2小时。然而，纤维可以被成像到后24小时的隔离。 </li><li>广场上共聚焦显微镜配有紫外激光器和60X / 63X客观的玻璃底菜。 </li><li>找到一个纤维。确保光纤连接牢固的菜通过点击显微镜基地。排除了被损坏的分解过程中的纤维。受损的纤维可以包含膜泡，吸收高水平的FM染料，卷曲或弯曲强烈。 </li><li>为了诱导的纤维膜损伤，在成像窗口有明确的核场的中心位置肌膜。专注于使用FM染料通道，使肌纤维膜是极其尖锐的肌膜。 </li><li>广场上利用FM 4-64通道肌膜的投资回报率十字线。 </li><li>照射在80％功率的1个像素点，3秒（〜350纳米x 350纳米范围）。调频染料会进入细胞在损伤部位。功率和时间可以调整，以增加或减少损伤的面积。 </li><li>损坏之前捕获光纤的图像，在损坏的时间，每2秒后的损害，那么，每10秒，至多5分钟。定时可以根据需要进行调整。单个图像可以被转换为时间推移电影在图像J。 </li><li>重复步骤3.3通过3.7每个培养皿最多3纤维。 </li></ol>

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

有没有利益冲突。

利用电穿孔来研究荧光标记的蛋白质在体内非常适合于肌肉由于缺乏合适的细胞系模型，忠实复制肌的整个细胞结构的研究。这个协议描述电穿孔和高分辨率共焦显微镜的利用率，激光伤人后提供蛋白定位和易位的详细成像。这种方法可以适用于研究其他细胞过程，包括细胞骨架重组，贩运和融合。 使用该协议，高达90％的FDB肌束明示质粒内的肌纤维，这可以容易地通过荧光显微镜之前和肌纤维离解后可以看到。有典型的表达与高表达观察更靠近注射部位的梯度。因为表达的梯度，变化的蛋白表达的效果可以来回监视马单一的实验。如同所有的转染方法，该方法是通过与过表达问题的限制。值得注意的是，与高水平表达，蛋白质聚集可发生。在我们的经验中，mCherry荧光标签表明比增强型绿色荧光蛋白（EGFP）标签降低分辨率和蛋白质结构域锐度多个聚集（例如，参见图4中的7）。此外，mTurquise2和金星荧光标记既表现出足够的荧光表达。然而，这两种蛋白质是比绿色荧光蛋白那么明亮。 这种方法可用于将图像的活的肌纤维在各种条件下，包括测试药剂和细胞伤人。我们使用的激光损伤扰乱肌膜，但其他创伤过程可适于此方法。为此，在形成膜的泡通过渗透休克以及诱导通过轧制玻璃珠也可以是膜损伤创造评估利用电肌纤维11,12。激光伤人，类型和激光的功率，以及目标将影响来创建膜病变13,14所需的时间长度。此外，外部缓冲器，缓冲器内钙的具体浓度和FM染料会影响修复过程13,15。这种方法已经过测试，在两个尼康和徕卡共聚焦成像系统，无一不是能够执行的技术。调频4-64是非常适合的实验与绿色荧光蛋白7。他人已经使用调频1-43，另一亲脂性染料结合带负在470nm和发射峰在610纳米，这限制了这种染料13,15,16的应用电荷的磷脂用激发峰。漂白和光毒性是由于过度成像两种可能的结果。确定曝光时间，图像频率和通道数之间的适当平衡成像很容易操纵，以减少这些影响的参数。

单个肌纤维比较大，高度组织化的合体细胞。有几个细胞培养模型为肌肉;然而，这些模型具有与它们不完全分化为成熟肌纤维的主要限制。例如，C2C12和L6细胞系来自于小鼠和大鼠，分别1-4。下血清饥饿的条件下，单核"成肌细胞样"细胞停止增殖，进行细胞周期撤出，并进入生肌程序与肌节形成多核细胞，称为肌管。由于长时间的培养条件，肌管可能会出现收缩特性和文化"抽搐"。人类细胞系现在也已被建立5。除了这些永生化细胞系，单核成肌细胞可以从肌肉和血清饥饿将形成肌管的类似的条件下进行分离。这些细胞系和原发性成肌细胞培养物是高度使用FUL因为它们可以转染的质粒或转导的病毒和用于研究细胞基本生物学过程。但是，这些细胞，即使当诱导以形成肌管，缺乏许多成熟肌肉组织的显着特征。具体地，肌管比单个成熟肌纤维小得多，缺乏肌纤维的正常形状。关键的是，肌管缺乏横（T-）管，需要有效的 Ca 2+整个myoplasm释放膜状网络。 的替代方法初级成肌或肌原细胞系造成把成熟的肌纤维。转基因可被用来建立标记蛋白质的表达，但这种方法是昂贵且耗时的。 在体内电穿孔小鼠肌肉的已作为其速度和可靠性6-10的优选方法。 方法用于体内电穿孔和的肌纤维公顷的有效隔离已经经过优化，鼠标趾短屈肌（FDB）肌肉6。该方法可以很容易地完成，是微创在体内表达由质粒诱导。现在这种做法是结合高分辨率成像方法，包括肌膜6,7激光破坏后成像。荧光染料和荧光标记的蛋白质的表达的组合可被用来监测在成熟肌纤维细胞的生物学过程。

<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.

电穿孔是一种微创技术，有效地引入了纯化的质粒DNA进趾短屈肌（FDB）肌束（图1A）。七天在转染后荧光标记的蛋白可视化隔离肌纤维（图1B）。分离的纤维然后铺板并制备用于成像上的共焦显微镜。纤维可以进行激光诱导的损伤。调频染料可用于识别的膜损伤的位点（ 图1C） 在消化过程中，少数肌纤维可能会受伤（白色箭头）（ 图2A）。受损的肌纤维可以通过膜起泡出席肌膜（黑色箭头）被识别。受损的纤维不适合于进一步的实验，不应该使用。代表共聚焦图像显示的表达Ø分离的肌纤维。图2B内发荧光融合蛋白。 CMV启动子的利用驱动器的最佳肌纤维蛋白的​​表达。 DIC成像显示了一个最佳的纤维。调频4-64是存在于该膜在较低的水平之前，损坏（图2C）。 装载了FM 4-64成像在Leica SP 5共聚焦显微镜肌纤维的形象代表。肌膜居中在视（白框）和ROI十字线（白色x）的领域上对齐肌膜的区域缺乏肌细胞核（图3，中）的脆，荧光边缘。核被避免，因为它们会影响FM染料分析后成像。经膜损伤，FM染料微创进入正常的肌纤维（ 图3右）。肌纤维有缺陷的膜修复将显示在激光损伤增加了FM染率水平。 -together.within页="1"&gt; <img alt="图1" src="/files/ftp_upload/53551/53551fig1.jpg" /> 图1.示意图 体内 电穿孔和激光损伤议定书 。（A）的透明质酸酶注射到麻醉小鼠的足垫。质粒DNA，然后注入并施加电压。 （ 乙 ）七天后喷射，所述趾短屈肌（FDB）束（白色箭头中间面板）被从足垫留下露筋（黑色箭头）分离。 （C）的纤维被镀和激光损伤的是活的肌纤维进行。左侧面板中，规模50微米。右侧面板中，规模5微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53551/53551fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 3551fig2.jpg"/&gt; 图2.隔离肌纤维表达荧光标记的蛋白质。七天电穿孔后，蛋白表达在整个检测到的肌纤维。 （A）分离过程中产生少量的不可用的肌纤维具有独特的膜起泡（黑色箭头）和膜破坏（白色箭头）的。比例尺为5μm。 （B）一种健康肌纤维的代表性的图像。均匀间隔的肌节可视化中的DIC成像（左）。一肌纤维表达膜联蛋白A6标记为绿色荧光蛋白（中图）。有最小的FM染料信号之前伤害（右图）。规模5微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53551/53551fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53551/53551fig3.jpg" /> 图3.对准激光在FM阳性肌膜十字星。的肌膜上感兴趣区域的视场范围内居中。肌膜被放大（白色虚线框）和投资回报率的十字线的肌膜在一个地区缺乏核（中图）的急剧FM-正沿对齐。一旦损坏，FM染料进入在损伤部位（右图，白色箭头）。规模5微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53551/53551fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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电穿孔，肌纤维的分离和成像

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

Nanyang Technological University

Research

JoVE Journal

Biology

12.2K 次观看.  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. 

视频: 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.

将人类成纤维细胞直接重新编程到肌细胞中，以研究神经肌肉疾病的治疗方法

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

科研

遗传学

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.

使用哺乳动物骨骼肌的DNA转染在体内电穿孔

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

生物学

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.

微创肌肉植入 (MIME)--一种促进供体细胞介导的肌的新实验技术

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

医学

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.

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

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.

从解离分析准备胚胎和幼体斑马鱼骨肌纤维

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).

单肌纤维隔离与文化从埃默里-德雷福斯肌肉萎缩的早期后纳塔尔发展穆林模型

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.

在成细胞分化和融合过程中利用活成像跟踪核

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.

使用纳米-牛顿分辨率力转换器从骨骼肌肉活检中隔离肌异物并确定收缩函数

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.

单一肌纤维培养测定法用于评估体外成人肌肉干细胞功能

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

可变长度小鼠肌肉纤维的酶促分离和表征

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.

作者聚焦：从小鼠后肢肌肉中分离长肌肉纤维，用于研究跨纤维类型的兴奋-收缩耦合

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).

通过酶解离小鼠六块后肢肌肉获得的完整短、中和长骨骼肌纤维：超越趾短屈肌

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