PDI CRISPR Activation Plasmid (h): sc-400676-ACT

Human P4HB encodes protein disulfide isomerase (PDI), a thiol–disulfide oxidoreductase and ER-resident chaperone that catalyzes disulfide bond formation, reduction, and isomerization to support oxidative protein folding. PDI is a core component of endoplasmic reticulum proteostasis networks, contributing to unfolded protein response signaling, ER-associated degradation, and quality control of secretory and membrane proteins. Through its roles in redox homeostasis and chaperone activity, PDI influences cellular responses to oxidative stress, hypoxia, and metabolic perturbation that intersect with inflammation and survival pathways. Dysregulated P4HB/PDI activity and elevated expression are frequently studied in contexts of proteotoxic stress and tumor biology, as well as in neurodegeneration and fibrotic remodeling where ER stress pathways are implicated.

PDI CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous P4HB expression without altering the underlying DNA sequence.

PDI CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the P4HB 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 P4HB transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous PDI expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native P4HB locus and enabling the study of PDI-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of PDI pathway restoration in tumor cells with silenced or reduced P4HB expression.

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