这项工作是由保罗·韦尔斯通合作研究中心（AR052646）。

所有的程序进行了审查和批准由美国宾夕法尼亚IACUC大学。 1。 EDL的剖析和准备（约30分钟） <ol><li>麻醉小鼠，以确保他们的经验在手术过程中没有疼痛或痛苦，但肌肉保持含氧的血液循环。我们经常使用氯胺酮/甲苯​​噻嗪通过IP鸡尾酒（100/10毫克/公斤）注射。手术的麻醉是由完全没有的踏板或palpedral的戒断反应能力，或缺乏的耳朵抽搐反应。 </li><li>固定用医用胶带的后肢，并去除皮肤低级前后肢暴露这个区域的肌肉。保持肌肉湿润以规则的时间间隔与该应用程序的PBS。 </li><li>在解剖的范围，作一小切口，以揭露的EDL肌肌腱近端外侧，膝盖。在该地区有两条肌腱，双方应削减到重新的EDL的moval。 </li><li>在脚踝内侧，切的TA肌肉肌腱暴露的EDL，沿元跗骨远端肌腱的。提起TA肌肉的方式，小心不要划伤或触摸EDL下。返回到远端肌腱的EDL，剪断每一个和吸引他们的脚踝，然后抓筋，轻轻将EDL远离其余的肢体。它应该释放从近端自由，但如果没有，返回的横向切口，以确保被切断肌腱。取下的肌肉和地方充满了冷冻含氧林格在解剖盘。引脚通过腱的肌肉，约休息的长度，这是在体内发现的长度。如果肌肉拉解剖销的长度太短时，肌肉是松弛的，在盘子里，又太长。 </li><li>安乐死鼠标紧随肌肉除去通过颈椎脱臼。 </li><li>配合缝合线的10笠头，尽可能接近的肌肉，但不触及肌肉。脚的肌肉使用的缝合线长度约休息。 </li></ol> 2。膜片夹层和准备（约30分钟） <ol><li>安乐死此过程之前的小鼠麻醉下，通过颈椎脱臼，如果相同的鼠标进行两个解剖，或由CO 2，然后通过确认颈脱位。 </li><li>在皮肤上做一个切口，暴露在腹腔和胸腔。打开腹腔，切肋骨下方的体壁。使用骨剪刀，膜片插入，切断围绕整个肋笼的肋线后，切穿脊椎以上开始。切血管贯穿中心的隔膜，使得隔膜可以容易地除去。 </li><li>取出膜片鼠标，并在解剖菜充满含氧林格的地方。轻轻搅动DIAPhragm的菜洗血。刷新林格液的需要。 </li><li>切一小条从中心腱膜片沿横向横膈膜肌收缩之情形的中央部中的纤维取向的肋。之间应该是2-4毫米宽的条带。使用骨剪刀，切割肋任一侧上的条带，留下约1-2毫米悬膜片条任一侧上的肋。 </li><li>配合中央肌腱缝合。扎缝合的横向凸肋端，然后把这些一起做了一大圈。 </li><li>两个条带，可以得到从任何给定的膜片（每侧一个从）。 </li></ol> 3。安装肌肉力学浴缸<ol><li>抓住缝线和使用这些附加的肌肉的一端上的刚性后，换能器的另一端上的力。 </li><li>调整长度，以确保肌肉不松弛，但不紧。一个很好的近似是休息MUSC乐长在1.4 - 1.5。 </li><li>浴应充满含氧林格液维持在22°C，延长肌肉的稳定性进行测试。肌肉应休息5分钟前在此沐浴功能测试，使肌肉温度平衡到22℃ </li></ol> 4。等长收缩<ol><li>建立超强刺激条件。后肌肉被放置在洗澡时，使用单0.5毫秒刺激脉冲产生抽搐，监控力输出。力逐渐增加电流，直到达到最高，但稳定的水平。增加电流超过该水平的剩余的实验10％。 </li><li>建立最佳长度。使用等距抽搐刺激，逐渐调整长度的肌肉，直到获得一个最大的力。休息的肌肉每次收缩〜10秒之间。最佳肌肉长度（L O）时，抽搐力是最大的。记录肌肉长度（长度之间的myotendinous路口EDL和之间的中心腱myotendinous结和肌肉插入肋），使用游标卡尺。 </li><li>最大等长强直力量。刺激肌肉设定为L O一段500毫秒，0.5毫秒脉冲在一系列的超强刺激，在融合频率（频率力的关系（ 例如编号：8）从高原的高原EDL肌肉是通常达到120赫兹和100赫兹的膈肌。刺激相应的频率在3次刺激一阵阵的每一块肌肉之间的休息时间为5分钟。 </li></ol> 5。离心收缩<ol><li>允许休息5分钟之间进行等长收缩和离心收缩的肌肉。 </li><li>刺激肌肉等长，在80赫兹的最初的500毫秒，随后用10％L O伸展在最后200毫秒刺激（以拉伸速度为0.5 L O/秒）。 </li><li>重复刺激模式，用5分钟的休息之间的所需数量的离心收缩。 </li><li>测量每个拉伸前的时间段中的收缩力。计算之间的第一个和最后一个收缩力下降。 </li></ol> 6。肌肉去除的设备<ol><li>缩短肌肉长度的传感器可以温和去除肌肉和发布，并返回它与林​​格解剖盘。 </li><li>肌肉删除缝线。 </li><li>对于肌肉的EDL，吸干肌肉两次，然后称量之前，后续加工（ 如冻结，定影等）。这将是很重要的，用于计算的横截面面积，和特定的力。 </li><li>对于膈肌，浸泡在0.1％普施安橙，膜浸透染料为15-20分钟。这将提供一个索引的剥离损伤。 </li><li>解剖的肌肉远离骨性插入的，以及占RAL肌腱。这是必要的，以提供精确的肌肉重量。吸干以上称重前及随后编写。 </li><li>计算截面积（CSA）： CSA（2毫米）=质量（mg）/ [（L O毫米）*（L / L o）*（1.06毫克/毫米3） 其中L / Lo是纤维肌肉长度比（0.45 EDL，1.0为隔膜）5和1.06是肌肉的密度。 </li></ol>

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生产和免费使用这篇文章是由极光科学。

这篇文章的目的是提供指导进行隔离两块肌肉的小鼠的肌肉功能 - EDL和隔膜。评估能够洞察这些肌肉或肌肉疾病的治疗候选人是否是有益的。对于这两种肌肉，强大的数据获取的主要因素是一个干净的清扫。因此，实践和完善的初步分离步骤是必不可少的，然后再移动到功能测试。此外，建立正常的肌肉功能的基准进行比较之前营养不良的肌肉，或在治疗的关键。这将确保研究结果的能力进行实验的人所给予的高度可变性不受。膜浸透染料的使用可以帮助优化解剖制剂，孵化任何肌肉的溶液中，含有这样的染料将马克受损最严重的纤维，并且可以作为一个索引d的issection成功。在肌肉纤维受损的数目最小化，将有助于优化测量。夹层损坏的纤维的荧光比偏心收缩损坏更明亮，因此，如果将染料的力学过程期间也使用，人们可以使用的染料的强度来区分两种类型的伤害。 膜片带剖析将几乎总是有纤维损伤，仅仅是因为通过沿着纤维的长度切割，一些不可避免地浏览销毁。在这些受损的纤维上染率是强大的，而这些通常是受限制的外边缘的准备。平均来说，我们观察到的损坏的纤维是〜3纤维的宽度（〜120微米）的任一侧上的肌肉带的频带。如果损坏的频带包括超过15％的肌肉，然后将数据将被丢弃。隔膜准备，也有约束的最佳大小，功能的措施。我们已经发现，件d的的iaphragm，超过5毫米宽开始自己折叠起来，，由于中央肌腱领带的是只在一个点上。这将导致比力下降的准备。我们还发现，窄条也有较低的比力，我们相信这是因为解剖过程中损坏的纤维包括总纤维数的比例较大。例如，如果0.1毫米每一侧上被破坏，然后是5％（2x0.1月/ 4毫米钢带）的肌肉制剂没有贡献给力，而如果带是只有1毫米，然后20 ％的肌肉制剂被损坏。因此，无论是损坏的区域的宽度，以及在整个制备过程中的宽度是重要的因素来控制。 最大等长收缩张力的测量要求所有在肌肉的肌纤维受到刺激。由于有巨大的功能装置的组件中的变化，这是必须为每个单独的组确定了。浴的尺寸或类型的激励器，例如，可以影响的刺激的强度。抽搐刺激是一个合理的方法来确定超强刺激条件。一旦这是建立一个特定的设置和肌肉，它可以被用于随后的研究。 与此相反，需要测量的最佳长度的任何给定的肌肉为每个准备使用如上所述的迭代过程。这将确保厚，细肌丝的重叠是最佳的，测量的最大潜在力发电能力。替代程序可以依靠与肌肉条纹相关的衍射图案，但是，这需要额外的设备，这里不作说明。 我们经常使用500毫秒刺激的持续时间，在这一领域的其他研究者使用此参数范围的中间，落在了。虽然这可能会导致一些疲劳的肌肉在收缩，这是显而易见的通过“下垂”的最大力量生产，这本身可以丰富。比如，在疲劳的差可以由不同类型的疗法包括那些目标钙处理，来实现，因此，在主动收缩力的凹陷可以作为一个指数为改善。另外，力的损失可能表明，筋缝合不够严密，并，他们滑倒在收缩。肌肉必须从浴中取出，并缝线必须重新绑如果发生这种情况。我们还使用了一系列的3强直收缩，这有助于稳定评估的准备。同样，缝合单将导致损失之间的收缩力，需要缝合重新捆绑。大肌肉也可能会产生缺氧的核心，从而导致力损失的协议。肌肉的大小是一个限制因素进行孤立的肌肉功能测试，EDL质量大于20 mg的肌肉失去力量，每个合同TION，并不能支持灌流的浴中。隔膜条不遭受相同的并发症，因为他们很瘦，足有长期在洗澡的可行性。 可以用于其他肌肉孤立的肌肉功能，包括比目鱼肌，这是常用的，但这里不作说明。可以采用许多相同的程序的比目鱼的编制及功能测试。然而，主要的差别是在刺激频率，离心收缩的参数。为函数的比目鱼肌补充使用，其他两个的肌肉，因此它应被看作是一个“标准的包”用于评估营养不良的小鼠4，7的一部分。 离心收缩提供了一个收缩的脆弱性指数，重要的是要使用的协议，结果在温和的野生动物的肌肉力量损失和重大损失的力未处理的营养不良的肌肉，使有一个宽的动态​​范围进行比较。任何力量缺乏的损失在正常肌肉离心收缩是太温和了，没有足够的区分是没有有效的治疗方法，和那些真正有利的。然而，在正常肌肉离心收缩力大幅下降，可能是太伟大了，梳理出与疾病相关的差异。我们的协议EDL和隔膜采用了0.5桢/秒的拉伸率，产生10％的长度变化。我们通常执行5个离心收缩，从而导致正常肌肉的力量和显着的损失在营养不良的肌肉力量小的损失。当然，所有这些参数可以改变，以增加整体的长度的变化，拉伸率，或离心收缩的数量，以便区分患病和健康的肌肉之间的差异，以及对一个特定类型的突变的影响或t正在研究reatment 1。只要有一个明显的区别正常和病变的肌肉，然后有一个金标准，在治疗方面达到。 总之，此协议规定执行等长收缩和离心收缩的指导方针，并希望确定潜在的隐患时要避免设置此技术在实验室中。

肌肉功能的测量有助于评价潜在治疗肌肉病理，以及测定这种组织生理学机制。对于肌肉疾病，使用的小鼠模型已成为一个重要组成部分，为了解基因型和表型之间的联系，以及扩展知识转化为设计和测试的潜在疗法。肌营养不良症，尤其是依赖于小鼠，以评估这些代理商和建立的临床前数据，需要向前移动到患者的试验中。一个常见的​​测量结果使用孤立的肌肉功能来决定力量，这是适用于广泛的研究。另一项措施是利用偏心，或延长，收缩，以确定肌细胞膜的完整性，这是缺乏在杜氏肌营养不良症，这种疾病的小鼠模型（MDX）。因此，至关重要的是，这些类型的measurem已废除，灵敏度高，重现。 鼠标趾长伸肌（EDL）肌肉已被广泛使用，为孤立的肌肉功能，由于其理想的几何形状和大小，均匀的纤维取向和明确的肌腱，2，5，6，10，12。 EDL肌肉等角功能测量的方法中已经描述的前一JOVE刊物8，以及在对待NMD的SOP 1。我们扩大了这些方法的描述，包括等长收缩和离心收缩。 EDL，包括heighted退化/再生周期，并减少力输出的疾病的标志是很明显的。 鼠标隔膜具有最快速的肌肉萎缩症的病理发展相比其他肌肉鼠标11。按6月龄中，累积纤维化包括约50％的肌肉。这将导致在显著impaire研发力量输出11。因此，可以防止纤维化浸润的治疗剂可以在膜片有效地进行评估。 的抗肌萎缩蛋白的肌肉损失，导致脆性增加和提高收缩所有肌肉损伤9。因此，许多杜氏进行性肌营养不良症的治疗都是为了更换为抗肌萎缩蛋白。因此，分析，评估这些战略已成为必不可少的离心收缩，它可以区分正常和营养不良的肌肉，以及确定什么好处一种特定的战略保护营养不良的肌肉收缩损坏2，3，4， 12。此过程需要一个双模式的伺服电机，可以调制/记录长度和力，或迅速分开的力换能器的方法的调整长度。

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 在体外肌肉测试系统</td><td>极光科学</td><td> 1200A </td><td></td></tr><tr><td>解剖显微镜</td><td>徕卡</td><td> MZ6 </td><td></td></tr><tr><td> ACE光源</td><td>肖特-Fostec </td><td> A20500 </td><td></td></tr><tr><td>解剖剪</td><td>精细科学工具</td><td> 14060-11 </td><td></td></tr><tr><td>角度的解剖剪刀</td><td>精细科学工具</td><td> 15006-09 </td><td></td></tr><tr><td>手术刀手柄</td><td>精细科学工具</td><td> 10003-12 </td><td>备用解剖工具</td></tr><tr><td>弯曲的手术刀片＃12 </td><td>美术学工具</td><td> 10012-00 </td><td>备用解剖工具</td></tr><tr><td>骨性剪刀</td><td>精细科学工具</td><td> 16044-10 </td><td></td></tr><tr><td> S＆T缝合线栓钳</td><td>精细科学工具</td><td> 00272-13 </td><td></td></tr><tr><td>杜蒙角度的不锈钢镊子 - </td><td>精细科学工具</td><td> 11203-25 </td><td></td></tr><tr><td>编织丝线缝合大小6-0 </td><td>泰利福医疗</td><td> 07-30-10 </td><td></td></tr><tr><td>医用胶带</td><td> Transpore </td><td> 3M </td><td></td></tr><tr><td>氯胺酮盐酸盐100毫克/毫升</td><td> Hospira公司</td><td> NDC 0409-2051-05 </td><td>最终剂量为80毫克/公斤</td></tr><tr><td> TranquiVed注射液（甲苯噻嗪的100毫克/毫升） </td><td> Vedco </td><td> NDC 50989-234-11 </td><td>最终剂量是10毫克/千克</td></tr><tr><td>活性橙14 </td><td> Sigma-Aldrich公司</td><td> R-8254 </td><td></td></tr><tr><td> 林格液组件 </td><td></td><td></td><td>解决方案是用95％O 2和5％CO 2，最终的pH值7.4平衡气体</td></tr><tr><td>氯化钠</td><td> Sigma-Aldrich公司</td><td> S7653 </td><td>终浓度为118毫米</td></tr><tr><td>氯化钾</td><td> Fisher Scientific则</td><td> P217-3 </td><td>终浓度为4.7毫米</td></tr><tr><td>二水氯化钙</td><td> Fisher Scientific则</td><td> C79-500 </td><td>终浓度为2.5毫米</td></tr><tr><td>磷酸二氢钾</td><td> Fisher Scientific则</td><td> P-285 </td><td>终浓度为1.2毫米</td></tr><tr><td>硫酸镁</td><td> JT贝克</td><td> 2500-01 </td><td>终浓度为0.57毫米</td></tr><tr><td> 4 - （2 - 羟乙基）管razine-1-乙磺酸（HEPES） </td><td> Fisher Scientific则</td><td> BP310-500 </td><td>终浓度：5.95克/升</td></tr><tr><td>葡萄糖</td><td> Sigma-Aldrich公司</td><td> G8270 </td><td>终浓度为5.5毫米</td></tr></tbody></table>

isometric and eccentric force generation assessment of skeletal muscles isolated from murine models of muscular dystrophies

肌肉疾病的潜在疗法的评价是敏感的和可重复的生理评估肌肉功能。由于许多临床前试验，依靠这些疾病的小鼠模型，已成为孤立的肌肉功能的Go / NoGo决定的候选药物进入患者的标准之一。趾长伸肌（EDL）和膈肌功能测试，这是主要的肌肉利用这些研究，我们将展示准备。 EDL肌肉的几何形状是非常孤立的肌肉，有两个方便的筋，具有体积小，可以支持灌流在洗澡。的隔膜具有深刻的进步的病理，在营养不良的动物，可以作为一个平台，打击纤维化评估很多潜在的治疗，并促进肌纤维的稳定性。协议的例行试验，包括等距和偏心TRIC收缩，将被显示。等长收缩力的力量，提供评估和离心收缩，有助于评估肌膜稳定，这是打乱了多种类型的肌营养不良症。从野生型和营养不良的肌肉肌肉的预期结果之间的比较，也可提供。这些措施可以补充的形态和生化测定组织的平衡，以及整个动物的肌肉功能的评估。

图1中所示的预期值从12周龄的动物的EDL肌肉的等长收缩力。因为MDX肌肉表现出代偿性肥大，总强直收缩力可以在mdx肌肉相比，年龄匹配的野生型控制。但是，更合适的功能输出的措施是特定的力（ 图2），其中，力被归为横截面面积来计算这个值。具体的力依赖于固有的肌肉功能的能力，在营养不良的肌肉更明显的弱点时，绝对力量和横截面面积占。在我们的手中，EDL mdx小鼠肌肉产生约20 - 25％的力量比那些匹配的年龄从10 - 26周龄的野生型小鼠。 对于隔膜，只有特定的力为相关的比较，因为该制剂是一块肌肉取决于的DISSEction。比较野生型和MDX膈肌之间的比力反映在这个组织病理学的进步。等长收缩力输出逐渐减少（ 图3）随着年龄的增长，因此，6个月的年龄，mdx小鼠的膈肌产生不超过一半的年龄相匹配的野生型控制功能输出的隔膜条。 离心收缩隔膜测试的一个例子如图4所示。随着每个后续的离心收缩，力量输出逐渐减少从野生型（C57）和mdx小鼠隔膜条。但是，该损失的力量就更加突出了在mdx小鼠的肌肉样本，大概是从抗肌萎缩蛋白及其相关蛋白的情况下。 <img alt="图1" src="/files/ftp_upload/50036/50036fig1.jpg" /> 在野生型的等距强直收缩力和mdx EDL肌肉f 图1。<html> ROM 12周大的老鼠。绝对的力量可以高于年龄匹配的MDX EDL肌肉代偿性肥大。 <img alt="图2" src="/files/ftp_upload/50036/50036fig2.jpg" /> 图2。肌肉横截面面积归等距强直收缩力比力，并揭示了郁闷EDL肌肉功能的能力mdx小鼠较野生型小鼠。轨迹是从图1中的相同的小鼠。 <img alt="图3" src="/files/ftp_upload/50036/50036fig3.jpg" /> 图3。力发电能力的逐渐丧失，比力评估是最明显的在隔膜（红柱）mdx小鼠相比，野生型（C57在蓝线）。 36/50036fig4.jpg“/&gt; 图4。武力作为一个离心收缩功能的数量从12周龄C57和mdx小鼠的膈肌的损失。数据平均值±SEM为N = 4每基因型的肌肉。

Watch this Scientific Journal Video about Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies at JoVE.com

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

肌肉功能的测量有助于肌肉病理评价的潜在疗法，以及测定这种组织生理学机制。我们将展示准备的伸趾长肌和膈肌功能测试。协议将显示为等长收缩和离心收缩，以及营养不良肌肉之间的业绩差异的一种病理状态，和野生的肌肉。

等距和骨骼肌产生偏心力评估隔离肌营养不良症的小鼠模型

Critical to the evaluation of potential  therapeutics for muscular disease are sensitive and reproducible physiological  assessments of muscle function. ...

isometric-eccentric-force-generation-assessment-skeletal-muscles

Research

JoVE Journal

Biology

32.5K 次观看.  School of Dental Medicine, University of Pennsylvania. 肌肉功能的测量有助于肌肉病理评价的潜在疗法，以及测定这种组织生理学机制。我们将展示准备的伸趾长肌和膈肌功能测试。协议将显示为等长收缩和离心收缩，以及营养不良肌肉之间的业绩差异的一种病理状态，和野生的肌肉。

视频: Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

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 ...

科研

生物学

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3&#039;UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

模仿人类小分子RNA的LightSwitch萤光素酶检测系统使用3&#39;UTR记者构造的microRNA识别目标在贴壁细胞

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

“体外骨骼肌微血管制备离体血管反应性的调查

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

体外评估对离体骨骼肌的收缩，疲劳性和交替“

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

钙火花的评估在正常肌肉纤维

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

组织分流和冻结的骨骼肌疾病模型

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal ...

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 ...

Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been reported that skeletal muscle may increase the extraction of glucose from the blood by up to 50-fold during exercise compared to resting conditions. The increase in muscle glucose uptake during exercise and insulin stimulation is dependent on the translocation of glucose transporter 4 (GLUT4) from intracellular compartments to the muscle cell surface membrane, as well as phosphorylation of glucose to glucose-6-phosphate by hexokinase II. Isolation and incubation of mouse muscles such as m. soleus and m. extensor digitorum longus (EDL) is an appropriate ex vivo model to study the effects of insulin and electrically-induced contraction (a model for exercise) on glucose uptake in mature skeletal muscle. Thus, the ex vivo model permits evaluation of muscle insulin sensitivity and makes it possible to match muscle force production during contraction ensuring uniform recruitment of muscle fibers during measurements of muscle glucose uptake. Moreover, the described model is suitable for pharmacological compound testing that may have an impact on muscle insulin sensitivity or may be of help when trying to delineate the regulatory complexity of skeletal muscle glucose uptake.
Here we describe and provide a detailed protocol on how to measure insulin- and contraction-stimulated glucose uptake in isolated and incubated soleus and EDL muscle preparations from mice using radiolabeled [3H]2-deoxy-D-glucose and [14C]mannitol as an extracellular marker. This allows accurate assessment of glucose uptake in mature skeletal muscle in the absence of confounding factors that may interfere in the intact animal model. In addition, we provide information on metabolic viability of incubated mouse skeletal muscle suggesting that the method&#160;applied possesses some caveats under certain conditions when studying muscle energy metabolism.

Intact regulation of muscle glucose uptake is important for maintaining whole body glucose homeostasis. This protocol presents assessment of insulin- and contraction-stimulated glucose uptake in isolated and incubated mature skeletal muscle when delineating the impact of various physiological interventions on whole body glucose metabolism.

测量小鼠分离和孵育成熟骨骼肌中胰岛素和收缩刺激的葡萄糖摄取

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been ...

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.

In this protocol, we describe how to generate murine primary epithelial colon monolayers directly from intestinal crypts. We provide experimental approaches to generate confluent monolayers on permeable filters, confluent monolayers for scratch wound healing and biochemical studies, and sparse and confluent monolayers for immunofluorescence analysis.

来自肠道密码的穆林原结肠上皮单层的生成

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in ...

In Vivo Measurement of Hindlimb Dorsiflexor Isometric Torque from Pig

Reliable assessment of skeletal muscle strength is arguably the most important outcome measure in neuromuscular and musculoskeletal disease and injury studies, particularly when evaluating regenerative therapies&#39; efficacy. Additionally, a critical aspect of translating many regenerative therapies is the demonstration of scalability and effectiveness in a large animal model. Various physiological preparations have been established to evaluate intrinsic muscle function properties in basic science studies, primarily in small animal models. The practices may be categorized as: in vitro (isolated fibers, fiber bundles, or whole muscle), in situ (muscle with intact vascularization and innervation but distal tendon attached to a force transducer), and in vivo (structures of the muscle or muscle unit remain intact). There are strengths and weaknesses to each of these preparations; however, a clear advantage of in vivo strength testing is the ability to perform repeated measurements in the same animal. Herein, the materials and methods to reliably assess isometric torque produced by the hindlimb dorsiflexor muscles in vivo in response to standard peroneal electrical stimulation in anesthetized pigs are presented.

In Vivo Measurement of Hindlimb Dorsiflexor Isometric Torque from Pig

The present protocol describes concise experimental details on the evaluation and interpretation of in vivo torque data obtained via electrical stimulation of the common peroneal nerve in anesthetized pigs.

体内 猪后肢背屈等距扭矩的测量

Reliable assessment of skeletal muscle strength is arguably the most important outcome measure in neuromuscular and musculoskeletal disease and injury ...