Core B - Transfection Core Laboratory

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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).