PHD3 CRISPR Activation Plasmid (m): sc-431083-ACT

Mouse Egln3 encodes prolyl hydroxylase domain-containing protein 3 (PHD3), an oxygen-sensing enzyme that hydroxylates HIF-α subunits and promotes their VHL-dependent ubiquitination and proteasomal turnover. Through regulation of HIF transcriptional programs, PHD3 influences hypoxia-responsive pathways controlling angiogenesis, metabolic adaptation, erythropoiesis, and inflammatory signaling. Egln3 activity also intersects with stress-response and apoptotic processes, contributing to cell fate decisions under nutrient or oxygen limitation. Dysregulated PHD3/HIF axis signaling is implicated in hypoxia-driven phenotypes relevant to tumor biology, ischemic injury models, and chronic inflammatory disease mechanisms, making Egln3 a useful node for pathway dissection in vivo and in cultured mouse cells.

HIF PHD3 CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Egln3 expression without altering the underlying DNA sequence.

HIF PHD3 CRISPR Activation Plasmid (m) 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.