在C2C12肌细胞造型强直性肌营养不良1
Summary
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。
Abstract
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。
Introduction
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'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'-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。
Protocol
Representative Results
Discussion
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'-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。
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Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| DMEM, high glucose | Life Technologies | 11965-084 | for culture medium |
| Fetal Bovine Serum – Premium | Atlanta Biologicals | S11150 | for culture medium |
| Penicillin-Streptomycin-Glutamine (100X) | Life Technologies | 10378-016 | for culture medium |
| Insulin from bovine pancreas | Sigma Aldrich | I6634-100MG | for differentiation medium |
| equine serum | Atlanta Biologicals | S12150 | for differentiation medium |
| FuGENE HD Transfection Reagent | Promega | E2311 | for transfection |
| G418 sulfate | Gold Biotechnology | G-418-10 | for drug resistant selection |
| Puromycin dihydrochloride | Sigma Aldrich | sc-108071 | for drug resistant selection |
| NuPAGE Novex 4-12% Bis-Tris Protein Gels, 1.0 mm, 15 well | Life Technologies | NP0323BOX | for western blot |
| NuPAGE Transfer Buffer (20X) | Life Technologies | NP00061 | for western blot |
| NuPAGE MES SDS Running Buffer (20X) | Life Technologies | NP0002 | for western blot |
| Amersham Protran Supported 0.2 NC, 300mmx4m | GE healthcare life science | 10600015 | for western blot |
| MF 20 | Developmental Hybridoma Bank | MF 20 | primary Ab for immunostaining |
| Goat anti-Mouse IgG (H+L) Secondary Antibody, Texas Red-X conjugate | Thermo Fisher Scientific | T-862 | secondary Ab for immunostaining |
| One step qRT-PCR MasterMix | AnaSpec | 05-QPRT-032X | for qRT-PCR |
| TriPure Isolation Reagent | Roche | 11667165001 | for RNA isolation |
| CUG-BP1 Antibody (3B1) | santa cruz | sc-20003 | primary Ab western blot |
| Actin Antibody | santa cruz | sc-1615 | goat polyclonal IgG for loading control |
| 293T Ecopack | Clontech | 631507 | cells for retrovirus preparation |
| pMSCV-puro | Clontech | 634401 | empty retroviral vector for retrovirus preparation |
| pMSCV-Celf1Flag-puro | house-constructed | not available | retroviral vector encoding Celf1Flag, used in retrovirus preparation |
| psPAX2 | gift from Didier Trono | not available | for lentivirus preparation |
| pMD2.G | gift from Didier Trono | not available | for lentivirus preparation |
| GFP-CUG5 | gift from M.S. Mahadevan | not available | details in reference 10 |
| GFP- CUG200 | gift from M.S. Mahadevan | not available | details in reference 10 |
| Triton X-100 | Sigma Aldrich | X100 | for immunostaining |
| paraformaldehyde | Sigma Aldrich | P6148 | for immunostaining |
| TWEEN 20 | Sigma Aldrich | P9416 | for immunostaining |
| DAPI | Sigma Aldrich | D9542 | for immunostaining |
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