IDH3A CRISPR Activation Plasmid (h): sc-404788-ACT

IDH3A encodes the alpha subunit of mitochondrial isocitrate dehydrogenase 3, an NAD+-dependent enzyme that catalyzes oxidative decarboxylation of isocitrate to α-ketoglutarate in the tricarboxylic acid (TCA) cycle. Through regulation of mitochondrial NADH production, IDH3A supports oxidative phosphorylation, redox balance, and metabolic coupling between glycolysis and biosynthetic pathways. Altered IDH3A activity and expression have been linked to metabolic reprogramming, mitochondrial dysfunction, and proliferative phenotypes, making it a relevant node for studying energy metabolism and stress responses. IDH3A-associated pathways intersect with hypoxia signaling, reactive oxygen species handling, and carbon flux into anaplerotic and epigenetic substrates derived from α-ketoglutarate.

IDH3A CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous IDH3A expression without altering the underlying DNA sequence.

IDH3A CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the IDH3A 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 IDH3A transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous IDH3A expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native IDH3A locus and enabling the study of IDH3A-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of IDH3A pathway restoration in tumor cells with silenced or reduced IDH3A expression.

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