γ-GCSm CRISPR Activation Plasmid (m): sc-420574-ACT

Gclm encodes the modifier subunit of glutamate-cysteine ligase (γ-GCSm), a key component of the rate-limiting step in glutathione (GSH) biosynthesis. By regulating catalytic efficiency and substrate affinity of the γ-glutamylcysteine ligase complex, γ-GCSm helps maintain intracellular redox balance and supports detoxification of electrophiles and reactive oxygen species. Gclm activity links to oxidative stress response networks including NRF2-dependent antioxidant programs, mitochondrial redox homeostasis, and xenobiotic metabolism. Altered Gclm/GSH capacity is widely used to model susceptibility to oxidative injury and redox-associated phenotypes relevant to neurodegeneration, metabolic dysfunction, inflammation, and toxicant responses in mouse systems.

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

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

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