HIF PHD3 CRISPR Activation Plasmid (h): sc-403671-ACT

EGLN3 encodes HIF prolyl hydroxylase 3 (PHD3), an oxygen-sensing dioxygenase that hydroxylates HIF-α subunits to promote VHL-dependent ubiquitination and proteasomal turnover under normoxia. By tuning HIF transcriptional output, PHD3 influences cellular adaptation to hypoxia, including glycolytic metabolism, angiogenic signaling, erythropoiesis-related programs, and oxidative stress responses. EGLN3 expression is dynamically regulated by hypoxia and other stress cues, linking it to feedback control within the HIF pathway and broader redox and mitochondrial homeostasis networks. Dysregulated EGLN3/PHD3 activity and HIF signaling are frequently studied in the context of tumor biology, ischemia-associated tissue stress, inflammation, and neurodegenerative processes, where oxygen-dependent transcriptional rewiring is a key driver of phenotype.

HIF PHD3 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous EGLN3 expression without altering the underlying DNA sequence.

HIF PHD3 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the EGLN3 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 EGLN3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous HIF PHD3 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native EGLN3 locus and enabling the study of HIF PHD3-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of HIF PHD3 pathway restoration in tumor cells with silenced or reduced EGLN3 expression.

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