Hemostasis, coagulation and maintenance of homeostasis involve expression of membranebound proteins such as tissue factor (TF) and protease-activated receptors. Spatiotemporal control of the gene expression of coagulation-related proteins such as TF may allow innovative experimental models of thrombosis and future translational applications. Here, we propose the use of a novel optogenetic gene expression system (EL222/C120) with rapid activation and deactivation kinetics to induce over-expression of coagulation-related proteins. EL222 is a bioengineered version of a bacterial light-oxygen voltage protein that acts as a transcriptional activator following illumination with blue light (450 nm). Following lightactivation, EL222 binds to C120 DNA binding / promotor elements. This system has a large dynamic range (>100-fold), rapid activation (<10 s) and rapid deactivation (>50 s) kinetics with a highly linear response to light. In Aim 1, we plan to achieve and optimize light-gated transcription of TF in endothelial cell (EC) lines transfected with tissue-specific vectors for EL222 as well as C120 / TATA box promotor elements upstream of cDNA for TF. We will add a ‘tagging sequence” to TF for monitoring of gene expression. In Aim 2, we intend to generate a knock-in mouse strain, in which TF expression in endothelial cells can be rapidly induced with linear kinetics by illumination with blue-light. We will use state-of-the art methodologies (e.g. TALEN/CRISPR) to insert the targeting vector in the mouse Rosa26 locus. To our knowledge, optogenetic switches have not yet been used in the field of thrombosis & hemostasis and such systems may prove to be innovative tools in translational hemostasis research.
06131 17-8277
