Transketolase CRISPR Activation Plasmid (h): sc-401941-ACT

Human TKT encodes transketolase, a thiamine pyrophosphate–dependent enzyme that catalyzes reversible two-carbon transfer reactions in the non-oxidative branch of the pentose phosphate pathway. By interconverting ribose-5-phosphate and glycolytic intermediates, transketolase helps coordinate nucleotide biosynthesis, redox balance through coupling to NADPH-producing reactions, and overall carbon flux during proliferation and metabolic stress. TKT activity influences cellular responses to oxidative stress and nutrient availability, and altered pentose phosphate pathway regulation has been associated with metabolic remodeling observed across diverse disease contexts, including cancer biology and inherited metabolic disorders. As a central node connecting glycolysis and pentose metabolism, TKT is frequently studied in pathway-level analyses of biosynthetic demand, antioxidant capacity, and cell-state transitions.

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

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

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