PDK4 CRISPR Activation Plasmid (h): sc-401355-ACT

Human PDK4 encodes pyruvate dehydrogenase kinase 4, a mitochondrial kinase that phosphorylates and inhibits the pyruvate dehydrogenase complex to limit pyruvate entry into the TCA cycle and promote metabolic switching toward fatty acid oxidation. Its expression is dynamically regulated by fasting, glucocorticoids, and PPAR signaling, integrating nutrient sensing with mitochondrial bioenergetics and cellular redox balance. By modulating glycolytic flux versus oxidative metabolism, PDK4 influences insulin responsiveness and lipid handling in metabolically active tissues. Dysregulated PDK4 activity and transcriptional control have been linked to metabolic stress phenotypes and are frequently studied in contexts such as insulin resistance, steatosis, and altered tumor cell metabolic programming.

PDK4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous PDK4 expression without altering the underlying DNA sequence.

PDK4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the PDK4 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 PDK4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous PDK4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native PDK4 locus and enabling the study of PDK4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of PDK4 pathway restoration in tumor cells with silenced or reduced PDK4 expression.

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