PEPCK-C/PCK1 CRISPR Activation Plasmid (m): sc-422131-ACT

Mouse Pck1 encodes the cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C), a rate-limiting enzyme in gluconeogenesis that converts oxaloacetate to phosphoenolpyruvate and links TCA cycle intermediates to glucose production. PEPCK-C integrates hormonal and nutrient signaling to coordinate hepatic glucose output and broader carbon flux through glycolysis/gluconeogenesis and anaplerotic/cataplerotic pathways. Altered Pck1 regulation is frequently used as a molecular readout of metabolic adaptation, including fasting responses, insulin and glucagon signaling, and shifts in mitochondrial–cytosolic substrate cycling. Dysregulated Pck1 expression is relevant to metabolic phenotypes such as insulin resistance, hepatic steatosis, and impaired glucose homeostasis in mouse models.

PEPCK-C/PCK1 CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Pck1 expression without altering the underlying DNA sequence.

PEPCK-C/PCK1 CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Pck1 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 Pck1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous PEPCK-C/PCK1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Pck1 locus and enabling the study of PEPCK-C/PCK1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of PEPCK-C/PCK1 pathway restoration in tumor cells with silenced or reduced Pck1 expression.

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