DNA电穿孔，肌纤维的分离和成像
Summary
This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.
Abstract
Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.
Introduction
单个肌纤维比较大，高度组织化的合体细胞。有几个细胞培养模型为肌肉;然而，这些模型具有与它们不完全分化为成熟肌纤维的主要限制。例如，C2C12和L6细胞系来自于小鼠和大鼠，分别1-4。下血清饥饿的条件下，单核“成肌细胞样”细胞停止增殖，进行细胞周期撤出，并进入生肌程序与肌节形成多核细胞，称为肌管。由于长时间的培养条件，肌管可能会出现收缩特性和文化“抽搐”。人类细胞系现在也已被建立5。除了这些永生化细胞系，单核成肌细胞可以从肌肉和血清饥饿将形成肌管的类似的条件下进行分离。这些细胞系和原发性成肌细胞培养物是高度使用FUL因为它们可以转染的质粒或转导的病毒和用于研究细胞基本生物学过程。但是，这些细胞，即使当诱导以形成肌管，缺乏许多成熟肌肉组织的显着特征。具体地，肌管比单个成熟肌纤维小得多，缺乏肌纤维的正常形状。关键的是，肌管缺乏横（T-）管，需要有效的 Ca 2+整个myoplasm释放膜状网络。
的替代方法初级成肌或肌原细胞系造成把成熟的肌纤维。转基因可被用来建立标记蛋白质的表达，但这种方法是昂贵且耗时的。 在体内电穿孔小鼠肌肉的已作为其速度和可靠性6-10的优选方法。
方法用于体内电穿孔和的肌纤维公顷的有效隔离已经经过优化，鼠标趾短屈肌（FDB）肌肉6。该方法可以很容易地完成，是微创在体内表达由质粒诱导。现在这种做法是结合高分辨率成像方法，包括肌膜6,7激光破坏后成像。荧光染料和荧光标记的蛋白质的表达的组合可被用来监测在成熟肌纤维细胞的生物学过程。
Protocol
Representative Results
Discussion
利用电穿孔来研究荧光标记的蛋白质在体内非常适合于肌肉由于缺乏合适的细胞系模型，忠实复制肌的整个细胞结构的研究。这个协议描述电穿孔和高分辨率共焦显微镜的利用率，激光伤人后提供蛋白定位和易位的详细成像。这种方法可以适用于研究其他细胞过程，包括细胞骨架重组，贩运和融合。
使用该协议，高达90％的FDB肌束明示质粒内的肌纤维，这可以容易地通…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是支持的美国国立卫生研究院授予NS047726，NS072027和AR052646。
Materials
| DMEM+A2:D32D42A2:D31AA2:D29 | Life Technologies | 11995-073 | Dissection Media Dilution: 50ml +100g BSA |
| Collagenase, Type II | Life Technologies | 17101-015 | Fiber Digestion Dilution: 160mg / 4ml DMEM (stock 40mg/ml aliquot) |
| Falcon 12-well dishes | Fisher Scientific | 877229 | Fiber Digestion |
| Ringers Solution | Fiber Digestion and Imaging Dilution: 146 mM NaCl 5 mM KCl 2 mM CaCl2 1 mM MCl2 10mM HEPES pH7.4 in H2O |
||
| 1ml Pipettes | Axygen | T-1000-C-R | Digestion |
| Razor Blades | Personna | 94-120-71 | Digestion |
| Precision Glide 30G needle | BD Biosciences | 305128 | Dissection |
| 1x PBS (-/-) | Life Technologies | 14190-250 | Dissection |
| Sylgard 184 | Fisher Scientific | 50-366-794 | Dissection |
| Forceps | FST by Dumont | #5/45 | Dissection |
| Forceps | Roboz | RS-4913 | Dissection |
| Scissors | World Precision Instruments | 500260 | Dissection |
| BSA | Sigma | A7906 | Dissection media Dilution: 100mg / 50ml DMEM |
| Falcon 60mm dishes | Fisher Scientific | 08772B | Dissection- used to hold sylgard |
| Clay | Electroporation | ||
| Stimulator | Grass | s88x | Electroporation |
| Clamps | Radio Shack | 270-356 | Electroporation |
| Triadine | Aplicare | 82-220 | Electroporation |
| Precision Glide 27G needle | BD Biosciences | 305136 | Electroporation |
| Simulator | Grass | SIU-V | Electroporation |
| Gauze | Covidien | 2146 | Electroporation |
| Hyaluronidase | Sigma | H4272 | Injection Dilution: Add 160ul PBS (1X) to 40µl 5x stock |
| 1cc U-100 Insulin Syringe (28G1/2) | BD Biosciences | 329420 | Injection |
| Eppendorf Tubes | Fisher Scientific | 02-682-550 | Injection |
| 35mm glass bottom Microwell dish | MaTek | P35G-1.5-14-C | Microscopy |
| FM 4-64 | Life Technologies | T-13320 | Microscopy Dilution: Final 2.5 ug/ml |
| FM 1-43 | Life Technologies | T-35356 | Microscopy Dilution: Final 2.5 ug/ml |
| Endotoxinfree Plasmid Maxi Kit | Qiagen | 12362 | Plasmid Purification |
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