Prothrombin CRISPR Activation Plasmid (h): sc-400962-ACT

F2 encodes prothrombin, a vitamin K–dependent zymogen that is proteolytically converted to thrombin, a central serine protease in the coagulation cascade. Thrombin cleaves fibrinogen to fibrin, amplifies coagulation via activation of factors V, VIII, XI, and XIII, and couples hemostasis to cellular responses through protease-activated receptor (PAR) signaling in platelets and vascular cells. Regulation of prothrombin activation intersects with anticoagulant pathways including antithrombin and the protein C system, shaping thrombin generation kinetics and clot stability. Dysregulated F2 expression or activity is linked to coagulation imbalance and thrombotic phenotypes, supporting its use as a mechanistic node for studying hemostasis and inflammation-associated vascular biology.

Prothrombin CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous F2 expression without altering the underlying DNA sequence.

Prothrombin CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the F2 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 F2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Prothrombin expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native F2 locus and enabling the study of Prothrombin-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Prothrombin pathway restoration in tumor cells with silenced or reduced F2 expression.

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