PEPCK-M/PCK2 CRISPR Activation Plasmid (h): sc-400725-ACT

Human PCK2 encodes the mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK-M), a key gluconeogenic enzyme that converts oxaloacetate to phosphoenolpyruvate using GTP. By coupling mitochondrial anaplerosis/cataplerosis to cytosolic carbohydrate and lipid biosynthesis, PEPCK-M helps coordinate TCA cycle flux, redox balance, and nutrient-dependent metabolic flexibility. PCK2 activity is linked to central carbon metabolism and can influence pathways including gluconeogenesis, glyceroneogenesis, and amino acid-derived carbon utilization. Altered PCK2 expression has been associated with metabolic rewiring observed in disorders of glucose homeostasis and in tumor metabolism, making it a useful node for studying mitochondrial control of biosynthetic programs.

PEPCK-M/PCK2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous PCK2 expression without altering the underlying DNA sequence.

PEPCK-M/PCK2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the PCK2 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 PCK2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous PEPCK-M/PCK2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native PCK2 locus and enabling the study of PEPCK-M/PCK2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of PEPCK-M/PCK2 pathway restoration in tumor cells with silenced or reduced PCK2 expression.

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