PI-9 CRISPR Activation Plasmid (m): sc-423120-ACT

Mouse Serpinb9 encodes PI-9, an intracellular serine protease inhibitor that restrains granzyme B activity to modulate cytotoxic lymphocyte effector function and prevent protease-driven apoptosis in antigen-presenting and immune-privileged cells. By buffering granzyme B–mediated cleavage events, PI-9 contributes to regulation of caspase activation, inflammatory cell survival, and immune homeostasis during cell–cell cytotoxic interactions. Serpinb9 expression is linked to mechanisms of immune evasion and persistence in inflammatory microenvironments, making it relevant to studies of tumor immunobiology, autoimmunity, and transplantation biology. Its inducible regulation also connects to stress and cytokine-responsive transcriptional programs that shape immune cell differentiation and tissue-specific immune tolerance.

PI-9 CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Serpinb9 expression without altering the underlying DNA sequence.

PI-9 CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Serpinb9 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 Serpinb9 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous PI-9 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Serpinb9 locus and enabling the study of PI-9-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of PI-9 pathway restoration in tumor cells with silenced or reduced Serpinb9 expression.

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