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
Summary
我们提供一种协议，使用小发夹（sh）RNA在C2C12表细胞细胞细胞中稳稳地敲除编码细胞外基质（ECM）蛋白质的基因。以 ADAMTSL2 为例，我们描述了在 C2C12 霉细胞到霉细胞分化期间，mRNA、蛋白质和细胞水平的敲除效率验证方法。
Abstract
细胞外基质 （ECM） 蛋白质对骨骼肌发育和平衡至关重要。C2C12肌细胞中ECM蛋白基因编码的稳定敲除可用于研究这些蛋白质在骨骼肌发育中的作用。在这里，我们描述了一个协议，以耗尽ECM蛋白ADAMTSL2为例，使用小发夹（sh）RNA在C2C12细胞。在shRNA质粒转染后，使用紫霉素分批选择稳定细胞。我们进一步描述了这些细胞系的维持以及通过mRNA表达、蛋白质表达和C2C12分化进行的型皮分析。该方法的优点是，在细胞培养基中血清耗尽后，稳定C2C12敲除细胞的生成速度较快，C2C12细胞可可靠地分化成多核化月管。C2C12细胞的分化可以通过明亮的场显微镜和测量规范标记基因的表达水平，如肌D、肌激素或肌苷重链（MyHC）来监测，表明C2C12肌细胞分化到肌管的进展。与具有小干扰（si）RNA的基因的瞬时击倒不同，在C2C12分化期间或在核管成熟期间表达的基因可以通过生成稳定表达shRNA的C2C12细胞来更有效地靶向。该方法的局限性是敲除效率的可变性，这取决于使用基于CRISPR/Cas9的基因敲除策略可以克服的特定shRNA，以及应考虑的shRNA的潜在偏离目标效应。
Introduction
细胞外基质 （ECM） 蛋白质为所有组织提供结构支持，调解细胞-细胞通信，并确定细胞命运。因此，ECM的形成和动态重塑对于维持组织和器官平衡1，2至关重要。为ECM蛋白编码的几个基因中的病理变异导致肌肉骨骼疾病，其表型从肌肉萎缩症到伪造血症3，4。例如，ADAMTSL2中的致病变异导致极其罕见的肌肉骨骼紊乱，表现为假肌肉生成，即骨骼肌质量明显增加5。加上小鼠和人类的基因表达数据，这表明ADAMTSL2在骨骼肌发育或平衡6，7中的作用。
我们在这里描述的协议是为了研究ADAMTSL2在细胞培养环境中调节骨骼肌发育和/或平衡的机制而开发的。我们稳稳地击倒了鼠C2C12霉菌细胞系中的ADAMTSL2。C2C12肌细胞及其分化成肌管是骨骼肌分化和骨骼肌生物工程8、9的一种描述良好且广泛使用的细胞培养模型。C2C12细胞在血清戒断后经历明显的分化步骤，导致培养3~10天后形成多核化月管。通过测量不同标记基因（如肌D、肌激素或肌苷重链（MyHC）的mRNA水平，可以可靠地监测这些分化步骤。在C2C12细胞中产生稳定基因敲除的一个优点是，与小干扰（si）RNA实现的瞬时敲除相比，在C2C12分化后期表达的基因可以更有效地定向，这种核化通常持续5~7天，并受转染效率的影响。此处所述协议的第二个优点是使用紫霉素选择快速生成批次 C2C12 敲除细胞。替代方案，如CRISPR/Cas9介导的基因敲除或从人类或靶基因缺陷小鼠分离原发性骨骼肌细胞前体，在技术上更具挑战性，或需要分别提供患者肌肉活检或靶基因缺陷小鼠。然而，与其他基于细胞培养的方法类似，使用C2C12细胞作为骨骼肌细胞分化模型存在局限性，如细胞培养的二维（2D）性质和缺乏对维持未分化的骨骼肌前体细胞至关重要的体内微环境10。
Protocol
1. 从大肠杆菌制备shRNA疟原体DNA 产生携带shRNA质粒的克隆细菌菌落 从商业来源获得携带靶性shRNA质粒的大肠杆菌的甘油库存和控制质粒（材料表）。注：使用了三种不同的shRNA质粒，针对的鼠阿达姆茨尔2 mRNA的不同区域。一个 shRNA 被选定针对Adamtsl2的 3’未翻译区域 （3’UTR）， 以方便使用表达质粒编码重组全长 ADAMTSL2 或单个 ADAMTSL2 蛋白质域的…
Representative Results
由于有效消除非耐药细胞，即未转染的细胞（图1B），在转染后10~14天内即可选择抗紫霉素的C2C12。通常，超过80%的细胞从细胞培养皿分离，这些细胞在常规细胞维护期间被移除。抗普诺霉素的C2C12细胞表达控制（分散）shRNA保持主轴形状，在低细胞密度下，长长细胞形态，并能分化成月管。血清提取时的C2C12分化可以通过明场显微镜和肌管标记肌苷重链（MyHC）的?…
Discussion
我们在这里描述了C2C12异型细胞中ECM蛋白稳定敲除的协议，以及C2C12表细胞分化到近骨的表型分析。有几个因素决定实验的结果，需要仔细考虑。在增殖阶段维持C2C12细胞是使C2C12细胞保持在霉细胞前体状态的关键步骤。保持C2C12细胞持续分化成月管的能力取决于i）细胞的通过数，ii）在日常维护期间培养细胞的密度，以及iii）营养供应，需要频繁和定期补充细胞培养基11，12，13。…
Divulgations
The authors have nothing to disclose.
Acknowledgements
D.H. 由美国国家卫生研究院（国家关节炎和肌肉骨骼和皮肤病研究所，NIAMS，赠款编号AR070748）和种子资助从Leni & Peter W. 五月骨科，伊坎医学院西奈山
Materials
| Acetone | Fisher Chemical | 191784 | |
| Agar | Fisher Bioreagents | BP1423 | |
| Ampicillin | Fisher Bioreagents | BP1760-5 | |
| Automated cell counter Countesse II | Invitrogen | A27977 | |
| Bradford Reagent | Thermo Scientific | P4205987 | |
| C2C12 cells | ATCC | CRL-1772 | |
| Chamber slides | Invitrogen | C10283 | |
| Chloroform | Fisher Chemical | 183172 | |
| DMEM | GIBCO | 11965-092 | |
| DMSO | Fisher Bioreagents | BP231-100 | |
| DNase I (Amplification Grade) | Invitrogen | 18068015 | |
| Fetal bovine serum | VWR | 97068-085 | |
| GAPDH | EMD Millipore | MAB374 | |
| Glycine | VWR Life Sciences | 19C2656013 | |
| Goat-anti-mouse secondary antibody (IRDYE 800CW) | Li-Cor | C90130-02 | |
| Goat-anti mouse secondary antibody (Rhodamine-red) | Jackson Immune Research | 133389 | |
| HCl | Fisher Chemical | A144S | |
| Incubator (Shaker) | Denville Scientific Corporation | 1704N205BC105 | |
| Mercaptoethanol | Amresco, VWR Life Sciences | 2707C122 | |
| Midiprep plasmid extraction kit | Qiagen | 12643 | |
| Myosin 4 (myosin heavy chain) | Invitrogen | 14-6503-82 | |
| Mounting medium | Invitrogen | 2086310 | |
| NaCl | VWR Life Sciences | 241 | |
| non-ionic surfactant/detergent | VWR Life Sciences | 18D1856500 | |
| Paraformaldehyde | MP | 199983 | |
| PBS | Fisher Bioreagents | BP399-4 | |
| PEI | Polysciences | 23966-1 | |
| Penicillin/streptomycin antibiotics | GIBCO | 15140-122 | |
| Petridishes | Corning | 353003 | |
| Polypropylene tubes | Fisherbrand | 149569C | |
| Protease inhibitor cocktail tablets | Roche | 33576300 | |
| Puromycin | Fisher Scientific | BP2956100 | |
| PCR (Real Time) | Applied Biosystems | 4359284 | |
| Reaction tubes | Eppendorf | 22364111 | |
| Reverse Transcription Master Mix | Applied Biosystems | 4368814 | |
| RIPA buffer | Thermo Scientific | TK274910 | |
| sh control plasmid | Sigma-Aldrich | 07201820MN | |
| sh 3086 plasmid | Sigma-Aldrich | TRCN0000092578 | |
| sh 972 plasmid | Sigma-Aldrich | TRCN0000092579 | |
| sh 1977 plasmid | Sigma-Aldrich | TRCN0000092582 | |
| Spectrophotometer (Nanodrop) | Thermo Scientific | NanoDrop One C | |
| SYBR Green Reagent Master Mix | Applied Biosystems | 743566 | |
| Trichloroacetic acid | Acros Organics | 30145369 | |
| Trizol reagent | Ambion | 254707 | |
| Trypan blue | GIBCO | 15250-061 | |
| Tryptone | Fisher Bioreagents | BP1421 | |
| Trypsin EDTA 0.25% | Gibco-Life Technology Corporation | 2085459 | |
| Water (DEPC treated and nuclease free) | Fisher Bioreagents | 186163 | |
| Western blotting apparatus | Biorad | Mini Protean Tetra Cell | |
| Yeast extract | Fisher Bioreagents | BP1422 |
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Citer Cet Article
Taye, N., Stanley, S., Hubmacher, D. Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin (sh)RNA. J. Vis. Exp. (156), e60824, doi:10.3791/60824 (2020).