PP2Cκ CRISPR Activation Plasmid (m): sc-433975-ACT

Ppm1k encodes the mitochondrial phosphatase PP2Cκ, a Mg2+/Mn2+-dependent PP2C family enzyme that regulates oxidative metabolism by dephosphorylating key targets involved in branched-chain amino acid (BCAA) catabolism. In particular, PP2Cκ modulates activity of the branched-chain α-ketoacid dehydrogenase (BCKDH) complex, linking mitochondrial nutrient flux to cellular energy homeostasis. Through its effects on BCAA utilization and mitochondrial function, Ppm1k is studied in contexts of metabolic stress responses and tissue-specific bioenergetics, including models relevant to insulin sensitivity, adipose and muscle metabolism, and mitochondrial dysfunction-associated phenotypes. Dysregulation of this pathway is associated with altered amino acid homeostasis and metabolic disease-relevant signaling states in experimental systems.

PP2Cκ CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Ppm1k expression without altering the underlying DNA sequence.

PP2Cκ CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Ppm1k 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 Ppm1k transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous PP2Cκ expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Ppm1k locus and enabling the study of PP2Cκ-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of PP2Cκ pathway restoration in tumor cells with silenced or reduced Ppm1k expression.

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