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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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?...

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 border="0" cellpadding="0" cellspacing="0" width="804">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DMEM, high glucose</td>
			<td style="width:112px;">Life Technologies</td>
			<td style="width:151px;">11965-084</td>
			<td style="width:325px;">for culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fetal Bovine Serum - Premium</td>
			<td style="width:112px;">Atlanta Biologicals</td>
			<td style="width:151px;">S11150</td>
			<td>for culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Penicillin-Streptomycin-Glutamine (100X)</td>
			<td style="width:112px;">Life Technologies</td>
			<td style="width:151px;">10378-016</td>
			<td>for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Insulin from bovine pancreas</td>
			<td style="width:112px;">Sigma Aldrich</td>
			<td style="width:151px;">I6634-100MG</td>
			<td>for differentiation medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">equine serum</td>
			<td style="width:112px;">Atlanta Biologicals</td>
			<td style="width:151px;">S12150</td>
			<td>for differentiation medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FuGENE&#160;HD&#160;Transfection Reagent</td>
			<td style="width:112px;">Promega</td>
			<td style="width:151px;">E2311&#160;</td>
			<td>for transfection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">G418 sulfate&#160;</td>
			<td style="width:112px;">Gold Biotechnology&#160;</td>
			<td style="width:151px;">G-418-10</td>
			<td>for drug resistant selection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Puromycin dihydrochloride</td>
			<td style="width:112px;">Sigma Aldrich</td>
			<td style="width:151px;">sc-108071</td>
			<td>for drug resistant selection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NuPAGE Novex 4-12% Bis-Tris Protein Gels, 1.0 mm, 15 well</td>
			<td style="width:112px;">Life Technologies</td>
			<td style="width:151px;">NP0323BOX</td>
			<td>for western blot</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NuPAGE Transfer Buffer (20X)</td>
			<td style="width:112px;">Life Technologies</td>
			<td style="width:151px;">NP00061</td>
			<td>for western blot</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NuPAGE MES SDS Running Buffer (20X)</td>
			<td style="width:112px;">Life Technologies</td>
			<td style="width:151px;">NP0002</td>
			<td>for western blot</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Amersham Protran Supported 0.2 NC, 300mmx4m</td>
			<td style="width:112px;">GE healthcare life science</td>
			<td style="width:151px;">10600015</td>
			<td>for western blot</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">MF 20</td>
			<td style="width:112px;">Developmental Hybridoma Bank</td>
			<td style="width:151px;">MF 20</td>
			<td>primary Ab for immunostaining</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Goat anti-Mouse IgG (H+L) Secondary Antibody, Texas Red-X conjugate</td>
			<td style="width:112px;">Thermo Fisher Scientific</td>
			<td style="width:151px;">T-862</td>
			<td>secondary Ab for immunostaining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">One step qRT-PCR MasterMix</td>
			<td style="width:112px;">AnaSpec</td>
			<td style="width:151px;">05-QPRT-032X</td>
			<td>for qRT-PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TriPure Isolation Reagent</td>
			<td style="width:112px;">Roche</td>
			<td style="width:151px;">11667165001</td>
			<td>for RNA isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CUG-BP1 Antibody (3B1)</td>
			<td style="width:112px;">santa cruz</td>
			<td style="width:151px;">sc-20003</td>
			<td>primary Ab western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Actin Antibody</td>
			<td style="width:112px;">santa cruz</td>
			<td style="width:151px;">sc-1615</td>
			<td>goat polyclonal IgG for loading control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">293T Ecopack</td>
			<td style="width:112px;">Clontech</td>
			<td style="width:151px;">631507</td>
			<td>cells for retrovirus preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pMSCV-puro</td>
			<td style="width:112px;">Clontech</td>
			<td style="width:151px;">634401</td>
			<td>empty retroviral vector for retrovirus preparation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">pMSCV-Celf1Flag-puro</td>
			<td style="width:112px;">house-constructed</td>
			<td style="width:151px;">not available</td>
			<td>retroviral vector encoding Celf1Flag, used in retrovirus preparation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">psPAX2</td>
			<td style="width:112px;">gift from Didier Trono</td>
			<td style="width:151px;">not available</td>
			<td>for lentivirus preparation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">pMD2.G</td>
			<td style="width:112px;">gift from Didier Trono</td>
			<td style="width:151px;">not available</td>
			<td>for lentivirus preparation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">GFP-CUG5</td>
			<td style="width:112px;">gift from M.S. Mahadevan</td>
			<td style="width:151px;">not available</td>
			<td>details in reference 10&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">GFP- CUG200</td>
			<td style="width:112px;">gift from M.S. Mahadevan</td>
			<td style="width:151px;">not available</td>
			<td>details in reference 10&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton&#160;X-100</td>
			<td style="width:112px;">Sigma Aldrich</td>
			<td style="width:151px;">X100</td>
			<td>for immunostaining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">paraformaldehyde</td>
			<td style="width:112px;">Sigma Aldrich</td>
			<td style="width:151px;">P6148</td>
			<td>for immunostaining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TWEEN&#160;20</td>
			<td style="width:112px;">Sigma Aldrich</td>
			<td style="width:151px;">P9416</td>
			<td>for immunostaining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DAPI</td>
			<td style="width:112px;">Sigma Aldrich</td>
			<td style="width:151px;">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?...

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

Universidad de Cartagena UDEC

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

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date1,2. Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies3. Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase4. The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours5. Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood6 as well as an ICH injection model of bacterial collagenase7, both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 &mu;l) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p&lt;0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.

在小鼠的脑出血模型:注射自体血液或细菌胶原酶

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

科研

医学

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.&#160;

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Models of spontaneous metastatic renal cell carcinoma (RCC) disease progression can be used for evaluating treatments in a clinically relevant setting. This protocol demonstrates different procedures for orthotopic kidney tumor cell implantation, proper nephrectomy, and finally outlines a necropsy guide for visual and bioluminescent scoring of metastatic burden and localization.

在小鼠模型自发转移性肾细胞癌（MRCC）肾切除

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

造型中风小鼠:在远端大脑中动脉永久性混凝

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with muscular dystrophy is an invaluable cellular source for in&#160;vitro disease model systems and can be used for drug screening studies. Patient-derived urine cells have been used in successful reprogramming into induced pluripotent stem cells in order to model dystrophic cardiomyopathy1. Addressing the safety concerns of integrating vector systems, we present a protocol using a non-integrating Sendai virus vector for transduction of Yamanaka factors into urine cells collected from patients with muscular dystrophy. This protocol generates fully reprogrammed clones within 2&#8211;3 weeks. The pluripotent cells are vector-free by passage-13. These dystrophic iPSCs can be differentiated into cardiomyocytes and used either to study disease mechanisms or for drug screening.

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

从生成性肌营养不良患者诱导多能干细胞的:尿源性细胞的有效整合，免费重编程

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Modeling Chemotherapy Resistant Leukemia In Vitro

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and progress in this site. The model presented in the current report utilizes human primary bone marrow stromal cells and osteoblasts as two representative cell types from the marrow niche that influence tumor cell phenotype. The in vitro co-culture conditions described for human leukemic cells with these primary niche components support the generation of a chemoresistant subpopulation of tumor cells that can be efficiently recovered from culture for analysis by diverse techniques. A strict feeding schedule to prevent nutrient fluxes followed by gel type 10 cross-linked dextran (G10) particles recovery of the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells (BMSC) or osteoblasts (OB) generating a "phase dim" (PD) population of tumor cells, provides a consistent source of purified therapy resistant leukemic cells. This clinically relevant population of tumor cells can be evaluated by standard methods to investigate apoptotic, metabolic, and cell cycle regulatory pathways as well as providing a more rigorous target in which to test novel therapeutic strategies prior to pre-clinical investigations targeted at minimal residual disease.

Modeling Chemotherapy Resistant Leukemia In Vitro

The current report summarizes a protocol that can be utilized to model the influence of the bone marrow microenvironment niche on leukemic cells with emphasis placed on enrichment of the most chemoresistant subpopulation.

建模化疗耐药白血病<em&gt;体外</em

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and ...

Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon skipping employs short DNA/RNA-like molecules called antisense oligonucleotides (AONs) that restore the reading frame and produce shorter but functional proteins. However, exon skipping therapy faces two major hurdles: limited applicability (up to only 13% of patients can be treated with a single AON drug), and uncertain function of truncated proteins. These issues were addressed with a cocktail AON approach. While approximately 70% of DMD patients can be treated by single exon skipping (all exons combined), one could potentially treat more than 90% of DMD patients if multiple exon skipping using cocktail antisense drugs can be realized. The canine X-linked muscular dystrophy (CXMD) dog model, whose phenotype&#160;is&#160;more similar to human DMD patients, was&#160;used to test the systemic efficacy and safety of multi-exon skipping of exons 6 and 8. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. In the current study, a cocktail of antisense morpholinos targeting exon 6 and exon 8 was designed and it restored dystrophin expression in body-wide skeletal muscles. Methods for transfection/injection of cocktail oligos and evaluation of the efficacy and safety of multi-exon skipping in the CXMD dog model are presented.

Exon skipping is currently a&#160;most promising therapeutic option for Duchenne muscular dystrophy (DMD). To expand the applicability for DMD patients and to optimize the stability/function of the resulting truncated dystrophin proteins, a multi-exon skipping approach using cocktail antisense oligonucleotides was developed and we demonstrated systemic dystrophin rescue in a dog model.

多外显子跳跃在犬X连锁肌营养不良用鸡尾酒反义寡核苷酸

Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

毛喉素诱导在肠组织体溶胀:一个<em&gt;体外</em&gt;检测方法的评估囊性纤维化患者对药物的反应

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts

Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide (AON)-mediated therapy. While SMN1 mutations are responsible for the disease, AONs targeting intronic splice silencer (ISS) sites in SMN2, including FDA-approved nusinersen, have been shown to restore SMN expression and ameliorate the symptoms. Currently, many studies involving AON therapy for SMA focus on investigating novel AON chemistries targeting SMN2 that may be more effective and less toxic than nusinersen. Here, we describe a protocol for in vitro evaluation of exon inclusion using lipotransfection of AONs followed by reverse transcription polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), and Western blotting. This method can be employed for various types of AON chemistries. Using this method, we demonstrate that AONs composed of alternating locked nucleic acids (LNAs) and DNA nucleotides (LNA/DNA mixmers) lead to efficient SMN2 exon inclusion and restoration of SMN protein at a very low concentration, and therefore, LNA/DNA mixmer-based antisense oligonucleotides may be an attractive therapeutic strategy to treat splicing defects caused by genetic diseases. The in vitro evaluation method described here is fast, easy, and sensitive enough for the testing of various novel AONs.

Various antisense oligonucleotides (AONs) have been shown to induce exon inclusion (splice modulation) and rescue SMN expression for spinal muscular atrophy (SMA). Here, we describe a protocol for AON lipotransfection to induce exon inclusion in the SMN2 gene and the evaluation methods to determine the efficacy in SMA patient fibroblasts.

剪接开关反义寡核苷酸诱导的 SMA 患者成纤维细胞外显子夹杂的评价

Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide ...

在C2C12肌细胞造型强直性肌营养不良1

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