HXK II CRISPR Activation Plasmid (m): sc-420866-ACT

Mouse Hk2 encodes hexokinase II (HXK II), a rate-limiting enzyme that phosphorylates glucose to glucose-6-phosphate at the entry point of glycolysis, thereby controlling carbon flux into energy production and biosynthetic pathways. HXK II function links glucose utilization to mitochondrial metabolism and can influence the balance between glycolysis and oxidative phosphorylation, with downstream effects on ATP generation, redox homeostasis, and anabolic precursor supply. In many cell types, HK2 expression is regulated by nutrient- and growth factor–responsive signaling networks, including PI3K–AKT and HIF-dependent programs that coordinate metabolic adaptation. Altered HK2 activity and expression are widely used as readouts of metabolic reprogramming relevant to proliferative and stress-adaptive states in models of inflammation, neurodegeneration, and tumor biology.

HXK II CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Hk2 expression without altering the underlying DNA sequence.

HXK II CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Hk2 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 Hk2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous HXK II expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Hk2 locus and enabling the study of HXK II-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of HXK II pathway restoration in tumor cells with silenced or reduced Hk2 expression.

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