DNA transfection of mammalian skeletal muscles using in vivo electroporation.
DNA transfection of mammalian skeletal muscles using in vivo electroporation.
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DOI：
10.3791/1520
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发表时间：
2009-10-19
期刊：
影响因子：
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通讯作者：
Vergara, Julio
中科院分区：
文献类型：
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作者：
DiFranco, Marino;Quinonez, Marbella;Vergara, Julio
细胞生物学中日益增长的兴趣是表达转基因修饰形式的必需蛋白（例如荧光标记的构建体和/或突变变体），以研究其内源性分布和功能相关性。在完全分化的细胞中实现这一目标的一种有趣的方法是通过各种方法将质粒体内转染到特定组织如肝脏、骨骼肌甚至大脑中。我们在这里提出了一个详细的描述，必须遵循的步骤，以有效地将遗传物质导入纤维的屈趾短肌（FDB）和骨间肌（IO）的成年小鼠使用体内电穿孔的方法。实验参数已被优化，以最大限度地增加转染的肌纤维的数量，同时最大限度地减少可能损害单个纤维中表达的蛋白质的质量和数量的组织损伤。我们已经证实，本文所述方法的实施导致可溶性蛋白质，即EGFP和ECFP、钙蛋白酶、FKBP 12、β 2a-DHPR等的高产率;结构蛋白质，即小型肌营养蛋白和α-辅肌动蛋白;和膜蛋白质，即α 1 s-DHPR、RyR 1、心脏Na/Ca（2+）交换器、NaV1.4 Na通道、SERCA 1等，当应用于小鼠和大鼠的FDB、IO和其它肌肉时。这些蛋白质中的一些的有效表达已被生物化学和功能证据所证实。然而，到目前为止，我们使用的最常见的验证方法是标准的荧光显微镜和双光子激光扫描显微镜（TPLSM），它允许识别不仅是整体表达，但也详细的细胞内定位，荧光标记的蛋白质结构。该方法同样可用于检测编码表达生理相关蛋白质的质粒（如此处所示），或检测旨在抑制正常表达蛋白质表达的干扰RNA（siRNA）（我们尚未测试）。应该注意的是，FDB和IO肌纤维的转染对于哺乳动物肌肉生理学的研究是特别相关的，因为从这些肌肉酶促解离的纤维目前是研究在电流或电压钳条件下兴奋性和兴奋-收缩偶联的基本机制的最合适的模型之一。
A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver, skeletal muscle, and even the brain. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP, calpain, FKBP12, beta2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and alpha-actinin; and membrane proteins, i.e. alpha1s-DHPR, RyR1, cardiac Na/Ca(2+) exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical and functional evidence. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions.