Core B - Transfection Core Laboratory

核心B - 转染核心实验室

• 批准号: 10117109
• 负责人: Victor Ruiz-Velasco
• 金额: $ 16.98万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10117109
• 关键词: ASIC channel; Acute; Afferent Neurons; Base Sequence; Chronic; Code; Complementary DNA; EP4 receptor; Electroporation; Elements; Exercise; Gastrocnemius Muscle; Genes; Hair; In Vitro; Individual; Inflammatory; Interleukin-6; Laboratories; Measures; Mediating; Messenger RNA; Microinjections; Muscle Form Glycogen Phosphorylase; Neurons; Nuclear; Organ; Peripheral arterial disease; Play; Productivity; Prostaglandins; Proteins; Receptor Signaling; Reflex action; Research Proposals; Reverse Transcriptase Polymerase Chain Reaction; Role; Skeletal Muscle; Small Interfering RNA; Spinal Ganglia; System; TNF gene; Techniques; Testing; Transfection; Ultrasonography; Western Blotting; Work; cytokine; design; experience; experimental study; image guided; in vivo; knock-down; lipofection; novel; programs; protein expression; response

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).

项目摘要/摘要 - 核心B：转染核心实验室 P01研究建议的一个重要目标是检查夸张的机制 运动压力反射（股骨阻塞后）受促炎性细胞因子（IL-6和TNF-）调节 α），NAV1.7和NAV1.8通道，嘌呤能（P2X3）通道，前列腺素（EP4）受体和酸传感器 在背根神经（DRG）神经元中表达的离子通道（ASIC）。对合同的回应 还将研究胃磷酸化酶急性敲低的传入术中的传入。 通过项目2和3，将定义这些渠道，接收器和信号元素的角色。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序列并提供为这些“短发”核苷酸编码的cDNA克隆 序列； 2）核心将进行体内和体外转染DRG（L4-L5）和胃肠痛 肌肉; 3）核心将使用定量RT-PCR（mRNA水平）和 蛋白质印迹（蛋白质定量）。这三个目标将帮助项目2和3测试其特定 假设。总体而言，这项工作将使人们更好地了解这些蛋白质在 夸张的运动训练反射在外周动脉疾病（PAD）中观察到。