PH-4 CRISPR Activation Plasmid (h): sc-409966-ACT

Human P4HTM encodes PH-4, an endoplasmic reticulum–associated prolyl 4-hydroxylase that catalyzes hydroxylation of proline residues on protein substrates, a post-translational modification that can influence protein folding, stability, and secretion. As part of the broader prolyl hydroxylase network, PH-4 links oxygen- and metabolite-dependent enzymatic activity to proteostasis and cellular adaptation pathways. Dysregulated hydroxylation chemistry and ER processing have been associated with altered stress signaling and metabolic phenotypes, supporting interest in P4HTM as a node connecting redox state, protein quality control, and cell-state regulation. Genetic variation and expression changes in P4HTM have been investigated in contexts involving metabolic and neurodevelopmental traits, motivating mechanistic studies in relevant human cell models.

PH-4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous P4HTM expression without altering the underlying DNA sequence.

PH-4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the P4HTM 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 P4HTM transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous PH-4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native P4HTM locus and enabling the study of PH-4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of PH-4 pathway restoration in tumor cells with silenced or reduced P4HTM expression.

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