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...

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+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.

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?...

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

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

Isolation of Genomic DNA from Mouse Tails

从鼠尾基因组DNA提取

科研

生物学

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.

电穿孔分枝杆菌

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.

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 ...

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.

颅面间质细胞的电

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.

二维和三维活细胞成像的DNA损伤反应蛋白

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.

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 ...

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.

结合单分子操纵和成像蛋白-DNA相互作用的研究

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.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

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.

样机小型化流式细胞仪及配套的混合微流控技术降低重力环境硬件演示

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