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Date of Graduation
Bachelor of Science (BS)
Department of Integrated Science and Technology
About one third of the world’s population is affected by hypertension, or high blood pressure, which increases an individual’s risk for cardiovascular disease. A major contributor to hypertension is dietary sodium intake. To assess an individual’s risk for hypertension, patients are put on a low sodium diet. However, research has shown that low salt intake can also have different and potentially harmful effects. Because of this, a genetic screen for salt sensitivity is needed to asses an individual’s salt sensitivity classification before testing and these results matched to a recommended dietary change. The kidney regulates the body’s fluid volume, so genetic factor within the renal system could be used for such a genetic screen. The dopaminergic system within renal proximal tubule cells is responsible for the majority of excretion and is highly influenced by a G-protein kinase, GRK4. The expression of GRK4, its association with the dopaminergic system, and the activity in varying salt sensitivity cell types can be indicators for salt sensitivity and provide a definitive diagnosis of the type of salt-related hypertension. Thus, a finely tuned treatment plan would be possible. To help study GRK4 expression, tricistronic lentivirus delivery systems were constructed to measure protein expression in vivo. To construct the vectors, scar-less DNA assembly methods allow for simpler and multipart construction, while web-based software j5 was developed to optimize cost, time efficiency, and reduce construction difficulty. Developing tricistronic vectors allows for introduction of a protein with two modes of measurement separated by 2A self-cleaving peptide sequences that impart protein autonomy. Bicistronic vectors, pLVX-GZ and pLVX-GC were constructed for utility and to establish a protocol for constructing tricistronic vectors. A tricistronic vector pLVX-GZC2 was been constructed containing GRK4, Zeocin resistance, and a fluorescence protein with P2A and T2A. Construction of such vectors will serve as a fast and cost-effective platform to fine-tune the differential diagnosis of salt-related hypertension.
Langouet-Astrie, Christophe, "Tricistronic Lentivirus vector construction using scar-less DNA assembly methods and web-based software j5 to help study GRK4" (2015). Senior Honors Projects, 2010-2019. 31.