Designing a vector system that works in hypoxic conditions by adding oxygen dependent degradation (ODD) domain to ptre / tet-off plasmid system and chacterization of the system in ischemic conditions

Tissue hypoxia, or ischemia, is the condition that describes the poor conveyance of oxygen and other vital products to tissues and organs. Ischemia progress silently, patients miss timely and proper treatments. In order to provide cardiovascular protection against ischemia and reperfusion, we are developing a vigilant gene therapy system whose expression level can be automatically up regulated by hypoxic ischemia. HIF-1α is a transcription factor that functions as a master regulator of oxygen homeostasis. HIF-1α protein levels increase under hypoxic conditions because of decreased O2 dependent prolyl-hydroxylation. ODD domain of HIF-1α was used as a switch to turn on the target gene expression in response to hypoxic signal. This hypoxia inducible plasmid system is a double plasmid system: transactivator plasmid and reporter plasmid. Chimeric transcription factor (TET-ODD) is being produced constantly from transactivator plasmid. In hypoxic condition, TET-ODD can stay stabilized in the cell and triggers expression of target gene. Our experiments showed that the ODD-mediated expression could quickly increase 4 fold within 6 hours of hypoxia (n=3) (p<0.05). Also, localization and stabilization of TET-ODD transcription factor was proved by immunofluorescence assay (IF) ad western blotting (WB) analysis. WB results showed that expression of TET-ODD protein increase 6-fold. The IF test showed that ODD linked tTA proteins were localized mainly in the cytoplasm. The ODD-TETR-VP16 protein showed high-ranking hydroxylation stability under hypoxia in protein modelling. From these results, we concluded that this hypoxia inducible double plasmid system could be used therapeutically to switch on genes that have proven beneficial effects in ischemia. Keywords: Gene Threapy, Hypoxia, Proline residue, Synthetic Biology, Transcription Factor