FN3K CRISPR Activation Plasmid (h): sc-412985-ACT

Human FN3K encodes fructosamine-3-kinase, a cytosolic enzyme that phosphorylates fructoselysine and related ketoamines on glycated proteins, promoting their destabilization and subsequent deglycation. By limiting accumulation of advanced glycation end products and glycation-mediated protein dysfunction, FN3K contributes to proteostasis and redox-sensitive metabolic homeostasis in contexts of elevated glucose flux. FN3K activity intersects with carbohydrate metabolism and cellular stress responses by modulating the burden of non-enzymatic protein glycation. Altered FN3K expression or function has been studied in relation to metabolic dysregulation and complications linked to chronic hyperglycemia, as well as broader effects on protein quality control.

FN3K CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous FN3K expression without altering the underlying DNA sequence.

FN3K CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the FN3K 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 FN3K transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous FN3K expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native FN3K locus and enabling the study of FN3K-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of FN3K pathway restoration in tumor cells with silenced or reduced FN3K expression.

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