MDH2 CRISPR Activation Plasmid (h): sc-402825-ACT

Human MDH2 encodes the mitochondrial malate dehydrogenase that catalyzes the reversible conversion of malate to oxaloacetate using NAD+/NADH, providing a key enzymatic step in the tricarboxylic acid (TCA) cycle. By linking mitochondrial redox balance to oxidative metabolism, MDH2 supports ATP production, anaplerotic flux, and metabolic coupling between glycolysis, the TCA cycle, and amino acid biosynthesis. MDH2 activity influences cellular responses to nutrient availability and mitochondrial stress, with downstream effects on reactive oxygen species handling and bioenergetic adaptation. Altered MDH2 expression or function has been associated with metabolic reprogramming observed in proliferative and neurodegenerative contexts, making it relevant for studies of mitochondrial dysfunction and redox-dependent signaling.

MDH2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous MDH2 expression without altering the underlying DNA sequence.

MDH2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the MDH2 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 MDH2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous MDH2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native MDH2 locus and enabling the study of MDH2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of MDH2 pathway restoration in tumor cells with silenced or reduced MDH2 expression.

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