Core B - Transfection Core Laboratory

项目总结/摘要-核心B：转移核心实验室 P01研究提案的一个重要目标是检查夸大的 运动加压反射（股动脉闭塞后）由促炎细胞因子（IL-6和TNF-α）调节。 α）、NaV1.7和NaV1.8通道、嘌呤能（P2 X3）通道、前列腺素（EP 4）受体和酸敏感 背根神经节（DRG）神经元表达的离子通道（ASIC）。收缩的反应 还将研究腓肠肌中肌磷酸化酶急性敲低后的传入。 通过项目2和3，这些通道，受体和信号元件的作用将被定义。在 为了实现这些目标，转染核心将通过三个目标提供关键帮助：1）核心 将设计有效的siRNA序列，并提供编码这些“短发”核苷酸的cDNA克隆 2）核心将进行DRG（L4-L5）和腓肠肌的体内和体外转染， 3）核心将采用定量RT-PCR（mRNA水平）验证蛋白质敲低， 蛋白质印迹法（蛋白质定量）。这三个目标将有助于项目2和项目3测试其具体的 假设总的来说，这项工作将有助于更好地理解这些蛋白质在细胞内的作用。 在外周动脉疾病（PAD）中观察到的过度运动升压反射。

PROJECT SUMMARY/ABSTRACT – CORE B: TRANSFECTION CORE LABORATORY An important objective of the P01 research proposal is to examine the mechanisms by which the exaggerated exercise pressor reflex (following femoral occlusion) is regulated by pro-inflammatory cytokines (IL-6 and TNF- α), NaV1.7 and NaV1.8 channels, purinergic (P2X3) channels, prostaglandin (EP4) receptors, and acid sensing ion channels (ASICs) expressed in dorsal root ganglion (DRG) neurons. The responses to contraction of afferents following acute knockdown of myophosphorylase in gastrocnemius muscle will also be investigated. Through Projects 2 and 3, the roles of these channels, receptors and signaling elements will be defined. In order to achieve these aims, the Transfection Core will provide key help through three objectives: 1) the Core will design effective siRNA sequences and provide cDNA clones that code for these `short hair' nucleotide sequences; 2) the Core will perform the in vivo and in vitro transfection of DRG (L4-L5) and gastrocnemius muscle; and 3) the Core will verify protein knockdown employing quantitative RT-PCR (mRNA levels) and Western blotting (protein quantification). These three aims will assist Projects 2 and 3 in testing their specific hypotheses. Overall, the work will lead to a better understanding of the role these proteins play in the exaggerated exercise pressor reflex observed in peripheral artery disease (PAD).