Tricistronic Lentivirus vector construction using scar-less DNA assembly methods and web-based software j5 to help study GRK4

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