Name: modeling myotonic dystrophy 1 in c2c12 myoblast cells
Uploaded: 2016-07-29T14:00:00.000+00:00
Duration: 9 min 39 s
Description: Watch this Scientific Journal Video about Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells at JoVE.com

We thank Drs. Tom Cooper from the Baylor College of Medicine, Mani S. Mahadevan from the University of Wisconsin-Madison, and Didier Trono from the University of Geneva for reagents. This work is supported by a University of Houston startup fund (YL), American Heart Association grant (YL, 11SDG5260033), and the National Natural Science Foundation of China (XP, 81460047).

1. C2C12细胞培养<ol><li>维持在生长培养基中100毫米板C2C12小鼠成肌细胞（Dulbecco改良的Eagle培养基（DMEM））补充有20％胎牛血清，100U / ml青霉素，100微克/ ml链霉素，和2mM L-谷氨酰胺。允许C2C12传代细胞变成约50 - 60％汇合。 </li><li>弃去生长培养基和洗涤C2C12细胞用3ml室温磷酸盐缓冲盐水（PBS）。除去PBS中并加入500微升的0.25％胰蛋白酶-EDTA分离​​细胞。放置板在37℃，5％CO 2培养箱3 - 5分钟。 </li><li>通过加入3ml生长培养基中和胰蛋白酶。吸管6 - 8倍以悬浮细胞。 1毫升悬浮的细胞的增加了100毫米的钢板，并在37℃，5％的CO 2下孵育。 </li></ol> 2. C2C12细胞转染/转导和选择<ol><li> C2C12质粒转<ol><li> 24小时在转染前，对晚7.0×10 5个的C2C12细胞成每个转染的六孔板的一个孔中。 注意:这导致50 - 由转染时60％汇合。 </li><li>允许转染试剂来在使用前室温。 </li><li>添加质粒DNA（GFP-CUG5或GFP-CUG200）2微克到200微升简要预热无血清培养基中并涡旋。添加6微升转染试剂的培养基，并使用涡旋立即混合30秒。孵育混合物，在室温下30分钟。 </li><li> C2C12细胞的媒体改变到2.5毫升的无血清生长培养基。转染混合物加入到细胞培养后。返回板，以5％的CO 2，37℃培养箱中。 </li><li>保持在转染混合物/无血清生长培养基将细胞4小时或最多过夜。更换介质回到生长培养基第二天或当细胞毒性是明显的。 注意:细胞毒性由CE的显微镜检查评估LLS。观察细胞形态和细胞分离的变化。只要没有明显的细胞毒性，在无血清生长培养基孵育过​​夜增强转染。 </li><li>选择的细胞在转染后48小时，加入1.2 - 1.6毫克/毫升的G418为约10天，直到对照培养（不含质粒转）没有活细胞。改变媒体每两天用补充有G418的生长培养基。 </li><li>维持在生长培养基中和5％的CO 2的G418抗性细胞在37℃，随后的实验。 </li></ol></li><li>逆转录病毒制剂和C2C12逆转录病毒转导<ol><li>通过用10％胎牛血清，100U / ml青霉素，100微克/ ml链霉素，和2mM L-谷氨酰胺补充的DMEM高糖制备HEK 293培养基中。 <ol><li>约24小时前转，板4.0×10 6嗜性为主HEK 293包装细胞100毫米板（转染80％融合）Containing细胞培养基。 </li></ol></li><li>允许转染试剂来在使用前室温。 </li><li>在1ml无血清生长培养基中混合15微克质粒DNA（的pMSCV-迪普罗或的pMSCV-Celf1Flag-迪普罗）。涡简要介绍。添加从步骤2.2.2 30微升转染试剂到DNA /无血清生长培养基管，并用涡流充分混合。在室温下孵育该混合物30分钟。 </li><li>转染混合物滴加加入到基于293亲嗜性的HEK包装细胞。轻轻地摇晃板，并返回他们回到5％的CO 2，37℃培养箱。 24小时后，介质改变至10ml预热的新鲜生长培养基。 </li><li>收获上清液（含逆转录病毒）使用移液管在转染后48小时，将上清液转移到50毫升离心管中。 10毫升新媒体热烈添加到平板，并返回到孵化器。保持上清液在4℃下。 注意:介质缓慢加入到t他阱的侧面，以便不破坏细胞单层。 </li><li>收获了第二批60上清液小时转染后，并与2.2.5上清池吧。离心机在500 xg离心，4℃7分钟以去除细胞碎片。 </li><li>将上清转移到新的50ml离心管中。使用逆转录病毒通过进到步骤2.2.9或等分试样并将上清液储存在-20℃以供将来使用转导C2C12细胞在这一点上。 </li><li>解冻从2.2.7逆转录病毒在室温下，如果在冻结的股票这里使用。 注意:避免多次冻融。 </li><li>板在转导瞄准30的同一天C2C12细胞 - 汇合的40％。 <ol><li>弃去生长培养基和洗涤C2C12细胞用3ml室温PBS中。除去PBS，加入500μl0.25％胰蛋白酶-EDTA，并在37℃，5％CO 2培养箱3孵育板- 5分钟。 </li><li>通过加入3ml生长MED中和胰蛋白酶IUM。吸管6 - 8倍以悬浮细胞。算使用血球细胞和8.0×10 5个 C2C12细胞添加到60毫米板。 </li></ol></li><li>将0.5ml的逆转录病毒感染细胞的一次60毫米的钢板在3毫升生长培养基。返回板的孵化器。 </li><li>感染后48小时 - 3辅以抗生素的选择（3微克/毫升嘌呤霉素1）毫升的新鲜介质替换。每天更换抗生素3毫升培养基〜5日，直至未转C2C12控制文化死了。 </li></ol></li><li>慢病毒编制和C2C12慢病毒转导 注:在生物安全水平执行所有的慢病毒有关的程序2柜，并按照生物安全准则。含有病毒废液必须在处置之前消毒。 <ol><li>板4.0×10 6个 293T细胞在100mm的板（〜在转染的当天80％汇合）用HEK 293细胞培养基（DMEM高葡萄糖，补充有10％胎牛血清，100U / ml青霉素，100微克/ ml链霉素，和2mM L-谷氨酰胺）转染前24小时。 </li><li>暖转染试剂至室温。混合慢病毒包装载体（4微克pMD2.G和6微克psPAX2）连同8微克慢病毒转移载体（Celf1的shRNA或加扰对照shRNA）的1ml无血清细胞培养基。涡简要介绍。 </li><li>添加36微升预热转染试剂与DNA / DMEM中管和通过涡旋混合均匀。在室温下孵育该混合物30分钟。 </li><li>从培养箱中取出293T细胞和转染混合物加入到板中。轻轻旋转盘，然后返回到5％的CO 2，37℃培养箱。 24小时后，更换用10ml新鲜培养基中的转染混合物。 </li><li>收集含上清液慢病毒48小时转染后，并将其转移到50毫升离心管中。加入10 mL新媒体热烈的板块，并将其返回孵化器。储存在4℃的上清液。 </li><li>收集第二批上清液60小时转染后，用上清从2.3.5相结合。在500 XG，4℃进行7分钟后短暂离心。 </li><li>将上清转移到新的50ml离心管中。如果使用新鲜的病毒，请继续步骤2.3.9。以供将来使用，含病毒的商店上清液在10毫升等份-20℃。 </li><li>在室温下在使用前从2.3.7解冻病毒，如果使用一个冷冻的储存。 注意:避免多次冻融。 </li><li>作为转导的同一天在2.2.9中描述板的C2C12-CUG200细胞（2.1节中获得）。 注:瞄准30 - 汇合的40％。 </li><li>将0.5ml病毒感染C2C12-CUG200的60毫米板与板返回孵化器。 </li><li>转导后72小时 - 用补充有选择抗生素（3微克/毫升嘌呤霉素1）的新鲜培养基替换培养基。文化传媒每天刷新的选择抗生素〜5日，直到所有未转染C2C12-CUG200控制文化死了。 </li><li>使用免疫印迹核实GFP-CUG200转染细胞Celf1击倒。 </li></ol></li></ol> 3. C2C12细胞分化<ol><li>板2×10 6个细胞在6孔板的一个孔在2.5ml生长培养基中24小时分化的开始之前。 注意:铺板密度可以被调整，使得在24小时达到满汇合。 <ol><li>冲洗融合细胞用PBS和加入2.5倍毫升低血清分化培养基（Dulbecco改进的补充有2％马血清的Eagle培养基，100U / ml青霉素，100微克/ ml链霉素，2mM的L-谷氨酰胺和1μM胰岛素） 。更换培养基每两天。 注意:保持股票胰岛素冻结，并在各媒体更改添加到媒体。 </li></ol></li><li>在C2C12分化，收集全样本titative RT-PCR（见第4节）在下列时间点:Day0，1，2，4，和6工艺在第6天免疫染色文化 - 8（见第5节）。 </li></ol> 4. RNA分离和定量RT-PCR <ol><li>使用制造商的方案进行RNA分离。分离后，悬浮在30微升不含RNA酶的水RNA沉淀和吸管上下几次。 注:样品可以进行到定量RT-PCR，也可以储存在-80℃。 </li><li>使用RT qPCR试 ​​剂盒定量RT-PCR 10和遵循制造商的指示。根据表1制备的反应混合物中。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 零件 </td><td> 体积（微升） </td><td> 终浓度 </td></tr><tr><td> 2X反应缓冲液</td><td> 10 </td><td> 1X </tD&gt; </tr><tr><td>正向引物</td><td> 0.5 </td><td> 200纳米</td></tr><tr><td>反向引物</td><td> 0.5 </td><td> 200纳米</td></tr><tr><td>探测</td><td> 0.5 </td><td> 200纳米</td></tr><tr><td>逆转录酶和RNA酶抑制剂</td><td> 0.1 </td><td> 0.25单位/毫升</td></tr><tr><td>模板</td><td> 1 </td><td> 100纳克总RNA </td></tr><tr><td>核糖核酸酶自由水</td><td> 7.4 </td><td> NA </td></tr><tr><td>总混音</td><td> 20 </td><td></td></tr></tbody></table> 表1.定量RT-PCR主混合物制备 <ol start="3"><li>运行使用温度曲线表2中的20微升RT-qPCR实验。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>逆转录步骤</td><td> 30分钟，48℃ </td></tr><tr><td> DNA聚合物酶的激活/逆转录酶失活</td><td> 10分钟，95℃ </td></tr><tr><td rowspan="2"> 40个循环</td><td> 15秒，95℃ </td></tr><tr><td> 1分钟，60℃下</td></tr></tbody></table> 表2.定量RT-PCR热分布 5.免疫染色<ol><li>固定单层培养在2.5ml新鲜制备的4％低聚甲醛/ PBS在室温下10分钟。 </li><li>透化的样品在2.5ml 0.1％的Triton X-100 / PBS在室温下30分钟。 </li><li>转移样品的潮湿室中。在2.5ml在37℃下进行30分钟封闭缓冲液（0.1％的非离子表面活性剂/ PBS，10％正常山羊血清）块中的样本。 </li><li>丢弃封闭缓冲液，并应用800微升初级抗体抗肌球蛋白重链在封闭缓冲液（1:100）稀释。孵育在室温下的潮湿室中过夜。 </li><li>洗板3恬ES用2.5ml洗涤缓冲液，第2天（0.1％聚山梨醇酯20 / PBS）（各10分钟）。 </li><li>除去洗涤缓冲液，并应用800微升二抗（山羊抗小鼠IgG，1:500在PBS中稀释）。孵育在湿润室中于室温下1小时。 </li><li>洗两次（每次10分钟）用2.5ml洗涤缓冲液。 </li><li>用800微升的DAPI（1微克/毫升，在蒸馏去离子水2 O稀释）5分钟孵育。 </li><li>用2.5ml洗涤缓冲液再次洗涤10分钟。 </li><li>改变洗涤缓冲液2.5毫升的PBS，并使用荧光显微镜来拍摄图像。 </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart-specific+overexpression+of+CUGBP1+reproduces+functional+and+molecular+abnormalities+of+myotonic+dystrophy+type+1.">Heart-specific overexpression of CUGBP1 reproduces functional and molecular abnormalities of myotonic dystrophy type 1.</a> Hum Mol Genet. 19 (6), 1066-1075 (2010).</li></ol>

The authors have nothing to disclose.

C2C12细胞系已被用作一个模型来研究肌形成11-14。这些细胞保留了成纤维细胞样外观，在含有20％胎牛血清媒体快速增殖和在含有2％马血清15媒体容易区分。快速生长和分化是在成肌细胞模型有利的特性。在这里，我们演示了如何使用质粒，逆转录病毒和慢病毒载体引进的cDNA，3&#39;-UTR，和shRNA进入C2C12细胞。转染/转导和分化维护一致性的关键点如下。 在每日细胞维持细胞密度是至关重要的。这些细胞需要低于50进行培养 - 70％汇合。由于短倍增时间，放置过夜时，高汇合（&gt; 70％）C2C12文化自发区别。而自发分化的细胞的分数可以是小的，存在的分化的细胞，将有助于在随后的分化实验不一致。具有低传代次数。此外，刚解冻的C2C12细胞是优选的。对于分化，细胞在预先确定的号码，这样他们在24小时后达到完全融合电镀。胰岛素是小的等分试样冷冻保存且每个分化培养基改变时被添加。这些措施提高分化的结果。 无血清培养基中增强C2C12细胞转染。该细胞可以在转染混合物/无血清培养基中保温过夜。在我们的实验室，我们实现了高达20％的转染率，伴随着细胞毒性轻微迹象。用于选择C2C12细胞的药物浓度较高，为1.2〜1.6毫克/毫升的G418或1至3微克/ ml嘌呤霉素，以及选择的持续时间比大多数其他细胞系更长。由于高药物浓度下，潜在的毒性和延长潜伏期，批与批在分化潜能的变化可能出现。关键的是要调整基于细胞形态的选择方案（浓度和持续时间）和治疗实验组和对照组在一起。 我们已经成功地推出了GFP-CUG200成再现DM1 3&#39;-UTR CUG扩展C2C12细胞。引入Celf1到C2C12细胞和Celf1的shRNA到GFP-CUG200 C2C12细胞已经允许通过Celf1上调和定位Celf1帮助DM1成肌细胞分化，分别探索触发细胞和分子事件的评价。总之，这里提出的成肌细胞C2C12受益模型强直性肌营养不良以及一般的肌肉细胞生物学和肌分化的调查。该模型的限制是，在细胞系中的结果可能不完全延伸到生物体。在当前的模型研究结果经常被提前到动物隔离新鲜的成肌细胞，并研究他们的不同点化。最终，我们证实在成肌细胞的生物研究结果。 本研究的独特优势是在C2C12细胞，这使得能够模拟在DM1顺序遗传/病态事件的多层基因操作。此前，DMPK CUG扩张的RNA毒性作用记录在成肌细胞11-14，而Celf1的病理作用在小鼠模型16,17分别研究。这些建模连续遗传/病理事件在技术上具有挑战性和时间，如果在动物模型中进行消费。如在本研究表明，联合使用荧光​​灯和耐药标记允许在强直性肌营养不良额外分子事件建模。本研究建立的模型是在解剖中DM1发病的详细机制有用的。他们也将是筛选针对DM1发病的不同步骤的药物价值。这些战略和本程序研究可能在成肌细胞建立其他肌肉疾病模型线索。

强直性肌营养不良（DM）是一种常染色体显性疾病，影响多个系统，最显着的心脏和骨骼肌1。有此疾病，DM1和DM2的两个亚型。 DM1是比较常见的，并且具有更严重的表现比DM2 2。遗传突变底层DM1是位于3&#39;非翻译区域的DM蛋白激酶基因（DMPK）3（UTR）的CUG三联体重复序列的扩张。在未受影响的个体的CUG重复次数变化从5到37相反，它增加了50多个，有时多达数千DM1患者4。其结果是，RNA结合蛋白，如的muscleblind样1（MBNL1），CUGBP和ELAV样家族1（Celf1），是misregulated。由于对扩大CUG重复封存，MBNL1失去其调节可变剪接5的能力。 Celf1，在另一方面，上调6,7。 Celf1的过度表达与肌肉相关的损失和虚弱，这是不归因于MBNL1功能丧失。动物模型模拟DM1相关改变，包括DMPK 3&#39;-UTR CUG扩张，MBNL1的损失，和Celf1的过表达，已经确立。然而，在成肌细胞建模DM1提供了一个有效的替代，特别是对于解剖DM1相关的细胞和分子事件。 C2C12成肌细胞系最初从受伤的C3H小鼠肌肉中分离，并广泛用于研究肌分化8,9。 C2C12细胞快速增殖中的胎牛血清（FBS）的含介质和容易当FBS的耗尽经历分化。然而，使用这种成肌细胞分化模型提出了两个挑战:C2C12细胞往往是基因转染/病毒转导性;并在细胞处理和分化过程的细微变化可导致肌管形成显着的变化。 我们的实验室经常使用C2C12细胞作为交流ELL模型，并开发了有效地提供质粒转染，逆转录病毒转导和慢病毒转基因导入C2C12细胞系10协议。在视频中，我们展示了用于转染/转导C2C12细胞并保持在建立DM1成肌细胞分化模型的一致性进行了优化程序。

<table><tbody><tr><td>DMEM, high glucose</td><td>Life Technologies</td><td>11965-084</td><td>for culture medium</td></tr><tr><td>Fetal Bovine Serum - Premium</td><td>Atlanta Biologicals</td><td>S11150</td><td>for culture medium</td></tr><tr><td>Penicillin-Streptomycin-Glutamine (100x)</td><td>Life Technologies</td><td>10378-016</td><td>for culture medium</td></tr><tr><td>Insulin from bovine pancreas</td><td>Sigma Aldrich</td><td>I6634-100MG</td><td>for differentiation medium</td></tr><tr><td>equine serum</td><td>Atlanta Biologicals</td><td>S12150</td><td>for differentiation medium</td></tr><tr><td>FuGENE&amp;nbsp;HD Transfection Reagent</td><td>Promega</td><td>E2311&amp;nbsp;</td><td>for transfection</td></tr><tr><td>G418 sulfate&amp;nbsp;</td><td>Gold Biotechnology&amp;nbsp;</td><td>G-418-10</td><td>for drug resistant selection</td></tr><tr><td>Puromycin dihydrochloride</td><td>Sigma Aldrich</td><td>sc-108071</td><td>for drug resistant selection</td></tr><tr><td>NuPAGE Novex 4 - 12% Bis-Tris Protein Gels, 1.0 mm, 15 well</td><td>Life Technologies</td><td>NP0323BOX</td><td>for western blot</td></tr><tr><td>NuPAGE Transfer Buffer (20x)</td><td>Life Technologies</td><td>NP00061</td><td>for western blot</td></tr><tr><td>NuPAGE MES SDS Running Buffer (20x)</td><td>Life Technologies</td><td>NP0002</td><td>for western blot</td></tr><tr><td>Amersham Protran Supported 0.2 NC, 300 mm x 4 m</td><td>GE healthcare life science</td><td>10600015</td><td>for western blot</td></tr><tr><td>MF 20</td><td>Developmental Hybridoma Bank</td><td>MF 20</td><td>primary Ab for immunostaining</td></tr><tr><td>Goat anti-Mouse IgG (H+L) Secondary Antibody, Texas Red-X conjugate</td><td>Thermo Fisher Scientific</td><td>T-862</td><td>secondary Ab for immunostaining</td></tr><tr><td>One step qRT-PCR MasterMix</td><td>AnaSpec</td><td>05-QPRT-032X</td><td>for qRT-PCR</td></tr><tr><td>TriPure Isolation Reagent</td><td>Roche</td><td>11667165001</td><td>for RNA isolation</td></tr><tr><td>CUG-BP1 Antibody (3B1)</td><td>santa cruz</td><td>sc-20003</td><td>primary Ab western blot</td></tr><tr><td>Actin Antibody</td><td>santa cruz</td><td>sc-1615</td><td>goat polyclonal IgG for loading control</td></tr><tr><td>293T Ecopack</td><td>Clontech</td><td>631507</td><td>cells for retrovirus preparation</td></tr><tr><td>pMSCV-puro</td><td>Clontech</td><td>634401</td><td>empty retroviral vector for retrovirus preparation</td></tr><tr><td>pMSCV-Celf1Flag-puro</td><td>house-constructed</td><td>not available</td><td>retroviral vector encoding Celf1Flag, used in retrovirus preparation</td></tr><tr><td>psPAX2</td><td>gift from Didier Trono</td><td>not available</td><td>for lentivirus preparation</td></tr><tr><td>pMD2.G</td><td>gift from Didier Trono</td><td>not available</td><td>for lentivirus preparation</td></tr><tr><td>GFP-CUG5</td><td>gift from M.S. Mahadevan</td><td>not available</td><td>details in reference 10&amp;nbsp;</td></tr><tr><td>GFP- CUG200</td><td>gift from M.S. Mahadevan</td><td>not available</td><td>details in reference 10&amp;nbsp;</td></tr><tr><td>Triton&amp;nbsp;X-100</td><td>Sigma Aldrich</td><td>X100</td><td>for immunostaining</td></tr><tr><td>paraformaldehyde</td><td>Sigma Aldrich</td><td>P6148</td><td>for immunostaining</td></tr><tr><td>TWEEN&amp;nbsp;20</td><td>Sigma Aldrich</td><td>P9416</td><td>for immunostaining</td></tr><tr><td>DAPI</td><td>Sigma Aldrich</td><td>D9542</td><td>for immunostaining</td></tr></tbody></table>

modeling myotonic dystrophy 1 in c2c12 myoblast cells

强直性肌营养不良1（DM1）是肌营养不良的一种常见形式。尽管一些动物模型已经建立了DM1，因为它们提供用于研究细胞和分子事件的有效的细胞替代成肌细胞模型仍然是重要的。虽然C2C12成肌细胞已被广泛用于研究肌形成，抗性基因转染或病毒转导，阻碍研究在C2C12细胞中。在这里，我们描述了一种优化的协议，包括日常维护，转染和转导过程的基因引入C2C12细胞和肌细胞分化的诱导。总的来说，这些程序使最佳的转染/转导效率，以及一致的分化的结果。在建立DM1成肌细胞模型将受益强直性肌营养不良的研究，以及其它肌肉疾病中描述的协议。

C2C12细胞与GFP-CUG5或GFP-CUG200染。耐药性选择后，稳定集合建立，其可以通过GFP表达（ 图1A）被可视化。在分化的成肌细胞肌管形成通过肌球蛋白重链的免疫染色10（ 图1B）进行检测。肌管形成的定量表明，融合指数从35.4±4.1％下降到2.6±1.1％和肌管领域，从35.6±2.2％的GFP CUG200（ 图1C）下降至2.7±0.8％由异常CUG扩张。的熔融指数和肌管领域经由核的由核的总数和总图像区域由肌管，分别覆盖的百分比除以肌管（≥2核）的总数进行计数。实时RT-PCR检测10被进行分析表达为一些肌肉不同的iation相关的基因，包括的MyoD，MyoG基因，MEF2C和Celf1。相比CUG5文化，CUG200在分化的开始增殖的成肌细胞增加Celf1 mRNA的表达。一致与以前的报告，Celf1上调与CUG-扩张肌营养不良1（ 图1D）有关。此外，Western印迹10一致显示Celf1蛋白水平升高（ 图1E），并CUG200分化（ 图1D）中抑制的MyoD，MyoG基因和MEF2C的表达。这些结果表明，该CUG膨胀导致肌管缺陷和受损成肌细胞分化。 研究Celf1在成肌细胞分化的作用，pMSCV- Celf1Flag逆转录病毒载体构建转导C2C12细胞。10嘌呤霉素-抗性克隆合并用于分化的研究。 Western blot检测结果表明，旗-TAgged Celf1只是在的pMSCV-Celf1Flag转导培养本和Celf1蛋白的表达上调（ 图2A）。相比于对照培养物（ 图2B），这表明Celf1的过表达严重损害成肌细胞分化时在Celf1过度表达细胞肌管的形成是罕见的。 要确定是否Celf1击倒在抢救CUG膨胀细胞分化不足，Celf1 shRNA的是由慢病毒载体运送到GFP-CUG200细胞。双抗性克隆（G418和嘌呤霉素）被选定并汇集了分化的研究。内源性Celf1蛋白的水平在Celf1的shRNA（ 图 3A）的存在显着降低。后分化6天，Celf1的shRNA对提高肌管形成的影响是明显的（ 图3B）。融合指数和肌管是如分别为16.1±3.0％和15.2±1.2％，相比4.6±0.8％，而在对照培养物（ 图3C）5.1±1.3％。同时，实时RT-PCR结果表明，MyoD的，MyoG基因和MEF2C的表达Celf1 shRNA的细胞（ 图3D）支承Celf1击倒救出肌细胞分化缺陷被显著增加。 <img alt="图1" src="/files/ftp_upload/54078/54078fig1.jpg" /> 图1:CUG-膨胀抑制心肌细胞分化的C2C12细胞。 （A）C2C12细胞中表达，选择后GFP-CUG5或GFP-CUG200无处不在。比例尺是100微米。这个数字已经从我们以前的纸10修改。（B）肌管形成于GFP-CUG5细胞，但在少GFP-CUG200细胞。肌管由肌球蛋白重链（MF-20）免疫染色可视化。比例尺是100微米。（C）的 </str翁&gt; GFP-CUG200文化显著降低熔融指数和肌管区。误差线代表标准偏差（D）Celf1的实时RT-PCR分析，转录成肌细胞分化过程中的因素的MyoD，MyoG基因，和MEF2C的表达。 mRNA水平后的GAPDH标准化作图。误差棒代表标准偏差。进行学生T检验，以测试比较对照。 N&gt; 3，* P &lt;0.05。（E）GFP-CUG200转增加Celf1蛋白的表达。 ß-actin的显示为装载控制。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54078/54078fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54078/54078fig2.jpg" /> 图2.表达Celf1削弱成肌细胞分化的。外源性Celf1Flag expre（一）证明裂变在C2C12细胞免疫印迹。（B）肌管形成在Celf1Flag过度表达的C2C12细胞受损。肌管是由MF-20染色可视化。比例尺是100微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54078/54078fig2large.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/54078/54078fig3.jpg" /> 图3. Celf1部分解救CUG -膨胀-诱导成肌细胞分化虚寒击倒。 （A）采用Western blot Celf1击倒的证明。（B）Celf1 shRNA的诱导肌小管在GFP-CUG200成肌细胞的形成。肌管是由MF-20染色可视化。比例尺是100微米。（℃）Celf1的shRNA救出细胞增加了熔融指数和肌管领域。误差棒代表标准偏差。（D）Celf1的实时RT-PCR分析，转录分化过程中MyoD的因素，MyoG基因和MEF2C的表达。 mRNA水平归到GAPDH的后作图。误差棒代表标准偏差。进行学生T检验，以测试比较的控制。 N&gt; 3，* P &lt;0.05。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54078/54078fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells at JoVE.com

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

在这个协议中，我们提出在建立强直性肌营养不良1成肌细胞模型，包括优化C2C12细胞维持，基因转染/转导，和肌细胞分化的步骤。

在C2C12肌细胞造型强直性肌营养不良1

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are ...

modeling-myotonic-dystrophy-1-in-c2c12-myoblast-cells

Nanyang Technological University

Research

JoVE Journal

Medicine

15.2K 次观看.  University of Houston. 在这个协议中，我们提出在建立强直性肌营养不良1成肌细胞模型，包括优化C2C12细胞维持，基因转染/转导，和肌细胞分化的步骤。 

视频: Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

工程骨骼肌组织中的小鼠成肌细胞的内皮祖细胞与应用电刺激

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

科研

生物工程

In Vitro Differentiation of Mature Myofibers for Live Imaging

Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily conserved structures that compose it are assembled remains under investigation. Their size and physiological features often constrain manipulation and imaging applications. The culture of immortalized cell lines is widely used, but it can only replicate the early steps of differentiation.
Here, we describe a protocol that enables easy genetic manipulation of myofibers originating from primary mouse myoblasts. After one week of differentiation, the myofibers display contractility, aligned sarcomeres and triads, as well as peripheral nuclei. The entire differentiation process can be followed by live imaging or immunofluorescence. This system combines the advantages of the existing ex vivo and in vitro protocols. The possibility of easy and efficient transfection as well as the ease of access to all differentiation stages broadens the potential applications. Myofibers can subsequently be used not only to address relevant developmental and cell biology questions, but also to reproduce muscle disease phenotypes for clinical applications.

In Vitro Differentiation of Mature Myofibers for Live Imaging

Muscle cells are among the most complex eukaryotic cells. We present a protocol for the in vitro differentiation of highly mature myofibers that allows for genetic manipulation and clear imaging during all developmental stages.

在成熟的肌纤维诱导分化实时成像

Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily ...

发育生物学

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

一个在培养皿中的功能电机组：脊髓植和肌肉细胞共培养

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

神经科学

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

目标移植的肌肉祖细胞的分子成像

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

医学

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin (sh)RNA

Extracellular matrix (ECM) proteins are crucial for skeletal muscle development and homeostasis. The stable knockdown of genes coding for ECM proteins in C2C12 myoblasts can be applied to study the role of these proteins in skeletal muscle development. Here, we describe a protocol to deplete the ECM protein ADAMTSL2 as an example, using small-hairpin (sh) RNA in C2C12 cells. Following transfection of shRNA plasmids, stable cells were batch-selected using puromycin. We further describe the maintenance of these cell lines and the phenotypic analysis via mRNA expression, protein expression, and C2C12 differentiation. The advantages of the method are the relatively fast generation of stable C2C12 knockdown cells and the reliable differentiation of C2C12 cells into multinucleated myotubes upon depletion of serum in the cell culture medium. Differentiation of C2C12 cells can be monitored by bright field microscopy and by measuring the expression levels of canonical marker genes, such as MyoD, myogenin, or myosin heavy chain (MyHC) indicating the progression of C2C12 myoblast differentiation into myotubes. In contrast to the transient knockdown of genes with small-interfering (si) RNA, genes that are expressed later during C2C12 differentiation or during myotube maturation can be targeted more efficiently by generating C2C12 cells that stably express shRNA. Limitations of the method are a variability in the knockdown efficiencies, depending on the specific shRNA that may be overcome by using gene knockout strategies based on CRISPR/Cas9, as well as potential off-target effects of the shRNA that should be considered.

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin shRNA

We provide a protocol to stably knock down genes encoding extracellular matrix (ECM) proteins in C2C12 myoblasts using small-hairpin (sh) RNA. Targeting ADAMTSL2 as an example, we describe the methods for the validation of the knockdown efficiency on the mRNA, protein, and cellular level during C2C12 myoblast to myotube differentiation.

使用小毛针（sh）RNA在C2C12霉细胞细胞系中编码细胞外基质蛋白的基因稳定击倒

Extracellular matrix (ECM) proteins are crucial for skeletal muscle development and homeostasis. The stable knockdown of genes coding for ECM proteins in ...

遗传学

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 ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

小鼠骨骼肌肉外泄原发性肌基器的分离和分化

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

从肌肉活检患者来源的细胞系疾病建模隔离和永生化

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, and spinal muscular atrophy, which are all associated with muscular atrophy. Primary cultures of spinal MNs have been used widely to demonstrate the involvement of specific genes in such diseases and characterize the cellular consequences of their mutations. This protocol models a primary MN culture derived from the seminal work of Henderson and colleagues more than twenty years ago. First, we detail a method of dissecting the anterior horns of the spinal cord from a mouse embryo and isolating the MNs from neighboring cells using a density gradient. Then, we present a new way of efficiently transfecting MNs with expression plasmids using magnetofection. Finally, we illustrate how to fix and immunostain primary MNs. Using neurofilament mutations that cause Charcot-Marie-Tooth disease type 2, this protocol demonstrates a qualitative approach to expressing proteins of interest and studying their involvement in MN growth, maintenance, and survival.

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

The goal of this technique is to prepare a highly enriched culture of primary motoneurons (MNs) from murine spinal cord. To evaluate the consequences of mutations causing MN diseases, we describe here the isolation of these isolated MNs and their transfection by magnetofection.

小鼠原代多元体的体外模拟？

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, ...

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 ...