TM CRISPR Activation Plasmid (h): sc-400956-ACT

THBD encodes thrombomodulin (TM), an endothelial cell surface glycoprotein that binds thrombin and shifts its substrate specificity to promote activation of protein C, linking coagulation control to anti-inflammatory signaling. TM participates in vascular homeostasis by modulating thrombin-mediated protease-activated receptor (PAR) pathways, limiting fibrin formation, and regulating endothelial barrier and leukocyte adhesion responses. Altered THBD expression or function is associated with dysregulated hemostasis and inflammatory vascular phenotypes, and it is frequently studied in contexts such as thrombosis susceptibility, sepsis-associated coagulopathy, and endothelial dysfunction. As a transcriptionally responsive endothelial marker, THBD also serves as a readout for shear stress, cytokine exposure, and vascular differentiation programs.

TM CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous THBD expression without altering the underlying DNA sequence.

TM CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the THBD locus in human cell lines. The system is built around a catalytically inactive Cas9 (dCas9) carrying two inactivating mutations (D10A and N863A) that eliminate nuclease activity while preserving DNA binding. This dCas9 is fused to VP64, a potent transcriptional activator, and is co-expressed with a blasticidin resistance gene for selection. The second plasmid encodes the MS2-p65-HSF1 fusion protein, a secondary activator complex that works in concert with dCas9-VP64, alongside a hygromycin resistance gene. The third plasmid encodes a target-specific 20 nt sgRNA fused to two MS2 RNA aptamers that recruit the MS2-p65-HSF1 complex to the activation site, accompanied by a puromycin resistance gene. The three plasmids are delivered at a 1:1:1 mass ratio for balanced expression of all system components.

Once assembled at the target locus, the SAM complex binds within approximately 200 bp upstream of the THBD transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous TM expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native THBD locus and enabling the study of TM-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of TM pathway restoration in tumor cells with silenced or reduced THBD expression.

For Research Use Only. Not Intended for Diagnostic or Therapeutic Use.