Prohibitin CRISPR Activation Plasmid (h): sc-416271-ACT

Human PHB encodes prohibitin, a conserved scaffold protein that localizes predominantly to the inner mitochondrial membrane and coordinates the stability of respiratory chain components with mitochondrial protein quality control. Prohibitin participates in regulation of mitochondrial morphology, membrane potential, and mitophagy, linking organelle fitness to cell-cycle progression, apoptosis, and cellular stress responses. Through interactions with signaling nodes such as MAPK and PI3K/AKT and modulation of transcriptional programs, PHB influences proliferation and differentiation in multiple cell types. Dysregulated PHB expression or function has been associated with altered metabolic homeostasis and tumor biology, making it a widely used marker and mechanistic entry point for studies of mitochondrial signaling and oncogenic pathways.

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

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

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