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：转基因核心实验室 P01研究提案的一个重要目标是检查夸张的 运动加压反射(股动脉闭塞后)受促炎细胞因子(IL-6和TNF-2)调节。 α)、NAV1.7和NAV1.8通道、嘌呤能(P2X3)通道、前列腺素(EP4)受体和酸觉 离子通道(ASICs)表达于背根神经节(DRG)神经元。对收缩的反应 我们还将调查腓肠肌中的肌磷酸酶在急性击倒后的传入。 通过项目2和项目3，将确定这些通道、受体和信号元件的作用。在……里面 为了实现这些目标，转基因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毛核苷酸的cDNA克隆 2)CORE将进行DRG(L4-L5)和腓肠肌的体内和体外转染 肌肉；以及3)核心将使用定量RT-PCR(信使核糖核酸水平)和 蛋白质印迹(蛋白质定量)。这三个目标将帮助项目2和3测试其特定的 假设。总体而言，这项工作将有助于更好地理解这些蛋白质在 在外周动脉疾病(PAD)中观察到夸大的运动加压反射。