IDH3G CRISPR Activation Plasmid (h): sc-404994-ACT

IDH3G encodes the gamma subunit of NAD-dependent isocitrate dehydrogenase 3 (IDH3), a mitochondrial matrix enzyme complex that catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate in the tricarboxylic acid (TCA) cycle. By contributing to NADH production, IDH3G supports mitochondrial respiration, redox balance, and metabolic flux that links carbohydrate catabolism to biosynthetic and signaling pathways. Altered regulation of TCA cycle enzymes can shift α-ketoglutarate availability and mitochondrial function, influencing stress responses and cellular differentiation states. Dysregulated mitochondrial metabolism and TCA cycle perturbations are recurrent features in cancer biology and other disorders characterized by impaired oxidative metabolism, making IDH3G a relevant target for mechanistic studies.

IDH3G CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous IDH3G expression without altering the underlying DNA sequence.

IDH3G CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the IDH3G 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 IDH3G transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous IDH3G expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native IDH3G locus and enabling the study of IDH3G-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of IDH3G pathway restoration in tumor cells with silenced or reduced IDH3G expression.

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