The Bioassay Core (Core C) will provide services to all three projects in this program project grant (PPG) and will be directed by Dr. Kathryn Sandberg. The overall goal of this core is to provide analytical support for each project of the PPG by aiding in gene silencing and gene expression through si_RNA and lentiviral strategies and to ensure quality control of employed strategies by genotyping knockout and transgenic (tg) animals and by measuring mRNA and protein expression of targeted genes of interest. Genotyping will be performed on single-gene deleted mice and at least two strains of transgenic mice bred in Core B including: gene deletions of extracellular superoxide dismutase (SOD-3);NADPH oxidase-4 (NOX-4);p47'"^?'', a critical subunit of NADPH oxidase-2 (p47'"'?'');ADP ribosyl cyclase 38 (CD38);fibroblast growth factor binding protein-1 (FGF-BP1) and -3 (FGF-BP3);dopamine-2 receptor (D2R);paraoxonase-2 (PON-2); overexpr-ession in vascular smooth muscle cells of p22''^?'*, a membrane subunit of NOX (VSMCP^^"'^"''tg) and catalase (VSMC^^'tg). This core will also determine levels of oxidative stress by measuring nitrates and nitrites, superoxide and the redox status of thiols by enzyme assays and capillary electrophoresis. Furthermore, this core is poised to develop new methods for gene manipulation for each project as gene manipulation technologies advance in the field.
Aim 1 will construct and develop gene silencing techniques including siRNA and lentiviral approaches to study the role of genes of interest both in vivo and in cell culture.
Aim 2 will assess gene expression at the DNA, mRNA and/or protein levels to ensure quality control of animal breeding performed in Core B and the efficacy of gene manipulation strategies in each of the three projects including via gene knockout, siRNA and shRNA-lentiviral approaches.
Aim 3 will determine the degree of oxidative stress by measuring nitrates, nitrites, reactive oxygen species (ROS) and the redox status of thiols in each of the three projects.
Hypertension is a global health concern. Fifty million Americans have hypertension that requires treatment and over 1 billion people woridwide have hypertension. In fact, suboptimal blood pressure is the number one risk factor for death throughout the worid. Though much research has been conducted on hypertension, the mechanisms underiying the main cause of hypertension, i.e., essential hypertension, remain unknown. This Core serves a crucial resource for this program project that is devoted to understanding the role of oxidative stress in hypertension.
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