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TRP X13 Inducible Over-Expression of Tissue Factor by a Novel Optogenetic System (EL222/C120)

 Funding period 01.07.2016 - 30.06.2017

Project Summary

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.

Principle Investigator

 Markus Bosmann, MD
Markus Bosmann, MD

Juniorgroup leader, Internist, Adjunct Assistant Professor of Molecular Medicine

Associate Professor of Medicine, Pathology & Laboratory Medicine, Boston University

06131 17-8277