PPOX CRISPR Activation Plasmid (h): sc-410546-ACT

Human PPOX (protoporphyrinogen oxidase) is a mitochondrial inner membrane enzyme that catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX, a key late step in heme biosynthesis. By controlling porphyrin flux, PPOX supports hemoprotein production and cellular redox balance, linking mitochondrial metabolism to oxygen handling and oxidative stress responses. Disruption or dysregulation of PPOX activity alters porphyrin homeostasis and is associated with porphyria phenotypes characterized by abnormal porphyrin accumulation. PPOX is therefore widely studied in pathways governing heme/porphyrin metabolism, mitochondrial function, and metabolic stress signaling.

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

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

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