PRODH CRISPR Activation Plasmid (h): sc-403063-ACT

Human PRODH encodes proline dehydrogenase (proline oxidase), a mitochondrial flavoprotein that catalyzes the first, rate-limiting step of proline catabolism by converting proline to Δ1-pyrroline-5-carboxylate, linking amino acid utilization to the TCA cycle and cellular redox balance. Through modulation of electron transfer to the respiratory chain, PRODH influences mitochondrial metabolism, reactive oxygen species signaling, and bioenergetic adaptation during nutrient stress. PRODH activity intersects with proline–P5C cycling and broader amino acid metabolism networks that affect apoptosis, autophagy, and oxidative stress responses. Dysregulation of PRODH has been associated with metabolic and neuropsychiatric phenotypes, and altered proline metabolism is frequently investigated in cancer metabolism and mitochondrial dysfunction models.

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

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

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