将质粒DNA电穿孔到小鼠骨骼肌中
Summary
将质粒DNA电穿孔到骨骼肌中是一种在不损害小鼠肌肉收缩力的情况下调节基因表达的可行方法。
Abstract
通过质粒电穿孔在小鼠骨骼肌中瞬时基因表达调节是评估正常和病理生理学的有用工具。靶基因的过表达或敲低使研究人员能够操纵单个分子事件，从而更好地了解影响肌肉质量，肌肉代谢和收缩力的机制。此外，编码荧光标签的DNA质粒的电穿孔使研究人员能够测量 体内骨骼肌中蛋白质亚细胞定位的变化。骨骼肌的关键功能评估包括肌肉收缩力的测量。在该协议中，我们证明在质粒DNA注射，电穿孔和基因表达调节后，整个肌肉收缩力研究仍然是可能的。该指导程序的目的是演示DNA质粒电穿孔到小鼠骨骼肌中的分步方法，以促进胫骨前伸肌肌的摄取和表达，以及证明骨骼肌收缩力不会受到注射和电穿孔的影响。
Introduction
体内骨骼肌质粒DNA电穿孔是通过调节各种生理和病理生理条件下的基因表达来评估骨骼肌生理学和分子信号传导变化的重要工具1，2，3，4，5，6，7，8，9.Wolff等人早在1990年就证明了向骨骼肌的实验性基因转移，其中RNA和DNA在没有电穿孔的情况下成功转移，并且荧光素酶表达维持至少2个月10。仅注射的转染效率相对较低是有问题的，爱原和宫崎骏在1998年通过将pCAGGS-IL-5构建体电穿孔到胫骨前肌中并测量血清IL-5表达11来证明电穿孔增加了基因转移。从那时起，许多研究已经研究了不同DNA浓度，体积和电穿孔参数的功效，以确保最大的基因转移效率。Mir等人测试了不同的电穿孔参数，包括电压，脉冲数，脉冲持续时间和频率，以及DNA浓度，并确定更大的电压，脉冲数和DNA浓度都有助于提高电穿孔效率12。高电穿孔电压的一个主要警告是，虽然它有助于增加对肌纤维的DNA摄取，但它也会导致肌肉损伤，从而混淆结果。Schertzer等人表明，在电穿孔后3天，200 V的肌电穿孔对约50%的肌纤维造成损害，而在50 V13下，只有10%的肌纤维受损。我们已经考虑了影响高效DNA转移与肌肉损伤的变量，发现每厘米卡钳宽度125 V的电压足以实现有效的基因转移。
电穿孔后肌肉纤维横截面积和整个肌肉收缩力的分析是测量由于基因调节引起的肌肉大小和功能变化的方法的重要方面。我们和其他人之前已经证明，单独对照载体的电穿孔不会导致肌纤维面积的减少。在这些研究中，绿色荧光蛋白（EGFP）构建体是DNA转染的有用荧光指示剂13，14。许多研究已经调查了电穿孔后TA的 原位 收缩力，并发现了不同的结果。一项研究表明，75 V/cm电穿孔在电穿孔后3天导致破伤风力降低约30%，到电穿孔后7天，破伤风力恢复到控制水平，而50 V/cm电穿孔不影响力13，15。另一项研究表明，在180 V / cm电穿孔后3小时，破伤风力损失了30%，7天后恢复到假力水平16。
在下面的详细程序中，我们演示了pcDNA3-EGFP质粒在小鼠的TA和长指伸肌（EDL）肌肉中的注射和电穿孔。我们还证明这种方法不影响EDL全肌收缩力。目的是证明质粒有效地摄取到肌纤维中，而不会引起功能丧失。
Protocol
所有使用动物的实验都是在宾夕法尼亚州立大学医学院进行的，该学院由宾夕法尼亚州立大学的机构动物护理和使用委员会批准，并按照1964年赫尔辛基宣言及其后来的修正案中规定的道德标准进行。使用12周龄雌性C57BL / 6小鼠进行该程序。在实验之前，所有手术工具都经过高压灭菌以进行无菌。 1. TA和EDL注射/电穿孔制备 注意：对于 TA 和 EDL 注…
Representative Results
电穿孔以促进骨骼肌中的基因转移是一种用于评估肌肉生理学变化的有用技术。我们已经展示了一个详细的分步程序，可以在TA和EDL肌肉中实现有效的基因转移。转染效率的差异是由许多变量引起的。这些变量包括电穿孔参数（脉冲，电压，脉冲持续时间等），基因构建体大小以及注入的DNA的浓度/体积。我们之前已经证明，5个脉冲在125 V/cm的电穿孔参数，20 ms持续时间间隔200 ms，足以在TA<sup class="…
Discussion
通过电穿孔增强的骨骼肌体内基因转移是调节肌肉中蛋白质表达的有用且相对简单的工具。我们已经展示了在EDL和TA肌肉中实现有效基因转移所需的步骤，并证明EDL的收缩性测量在手术后是可行的。该技术不需要更复杂的病毒载体，并允许比较单个肌肉中转染和非转染的肌肉纤维横截面积。该程序的局限性在于构建摄取效率不完整，并且一些肌纤维仍未转染。
所讨论的…
Divulgations
The authors have nothing to disclose.
Acknowledgements
没有
Materials
| 4-0 Nylon suture (non-absorbable) | Ethicon | 662G | Suture to close skin incision |
| 50µl Hamilton syringe | Hamilton | 80501 | microsyringe |
| C57BLl/6NHsd mice | Envigo | 044 | 12 week-old female mice used for experimentation |
| Caliper Electrode | BTX | 45-0102 | 1.0cm x 1.0cm stainless steel |
| Dynamic Muscle Control Data Acquisition/analysis | Aurora Scientific | 605A | Software used for muscle contractility measurement and analysis |
| ECM 830 Electroporation System | BTX | 45-0662 | electroporator |
| EndoFree Plasmid Maxi Kit | Qiagen | 12362 | Plasmid purification kit |
| Extra Narrow Scissors | Fine Science Tools | 14088-10 | Scissors for blunt dissection |
| Force Transducer | Aurora Scientific | 407A | To measure force from EDL |
| Micro-Masquito Hemastats | Fine Science Tools | 13010-12 | Hemastats for surturing |
| pcDNA3.1 mammalian expression vector | Fisher Scientific | V79020 | Control Vector |
| pcDNA3-EGFP expression plasmid | Addgene | 13031 | Plasmid for GFP expression |
| Semken curved forceps | Fine Science Tools | 11009-13 | Forceps for surgery |
| Surgical blades stainless steel no. 10 | Becton Dickinson | 37 1210 | Scalpel blades |
| Tissue-Tek O.C.T. media | VWR | 25608-930 | Freezing media for histology |
| Wheat Germ Agglutinin- Texas Red | Thermo-Fisher Scientific | W21405 | Membrane staining for muscle cross section |
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Citer Cet Article
Hain, B. A., Waning, D. L. Electroporation of Plasmid DNA into Mouse Skeletal Muscle. J. Vis. Exp. (182), e63916, doi:10.3791/63916 (2022).