PFKFB4 CRISPR Activation Plasmid (h): sc-410933-ACT

Human PFKFB4 encodes 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4, a bifunctional enzyme that controls cellular levels of fructose-2,6-bisphosphate and thereby tunes glycolytic flux through allosteric regulation of PFK1. By modulating carbon flow between glycolysis and associated metabolic outputs, PFKFB4 contributes to metabolic adaptation under stress conditions such as hypoxia and nutrient limitation, with downstream effects on redox balance and biosynthetic capacity. Altered PFKFB4 expression and activity have been linked to metabolic reprogramming observed across diverse disease-relevant contexts, particularly in models of proliferative signaling and microenvironmental stress. As a result, PFKFB4 is frequently studied in pathways connecting glycolysis, HIF-regulated responses, and growth control.

PFKFB4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous PFKFB4 expression without altering the underlying DNA sequence.

PFKFB4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the PFKFB4 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 PFKFB4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous PFKFB4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native PFKFB4 locus and enabling the study of PFKFB4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of PFKFB4 pathway restoration in tumor cells with silenced or reduced PFKFB4 expression.

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