EG-VEGF CRISPR Activation Plasmid (h): sc-404774-ACT

PROK1 encodes endocrine gland–derived VEGF (EG-VEGF), a secreted pro-angiogenic cytokine that signals primarily through the G protein–coupled receptors PROKR1 and PROKR2 to regulate endothelial cell migration, permeability, and vascular remodeling. EG-VEGF shows tissue-selective activity and contributes to endocrine and reproductive vascular specialization, linking extracellular cues to downstream MAPK/ERK, PI3K/AKT, and calcium-dependent signaling programs. In human biology, dysregulated PROK1 signaling has been associated with aberrant angiogenesis and inflammatory microenvironment changes, making it a useful node for studying vascular niche regulation. This pathway is frequently examined in contexts where endothelial–stromal communication and oxygen- or cytokine-responsive transcriptional programs influence tissue remodeling.

EG-VEGF CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous PROK1 expression without altering the underlying DNA sequence.

EG-VEGF CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the PROK1 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 PROK1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous EG-VEGF expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native PROK1 locus and enabling the study of EG-VEGF-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of EG-VEGF pathway restoration in tumor cells with silenced or reduced PROK1 expression.

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