γ-GCSc CRISPR Activation Plasmid (m): sc-420573-ACT

Mouse Gclc encodes the catalytic subunit of glutamate-cysteine ligase (γ-GCSc), the rate-limiting enzyme in glutathione (GSH) biosynthesis. By controlling cellular GSH pools, γ-GCSc regulates redox homeostasis, detoxification of electrophiles and xenobiotics, and antioxidant defense downstream of NRF2/KEAP1 signaling. Gclc activity influences mitochondrial function, inflammatory signaling, and susceptibility to oxidative stress across tissues, linking its dysregulation to pathways implicated in liver injury, neurodegeneration, metabolic dysfunction, and cancer-associated redox adaptation. As a central node in thiol metabolism, Gclc is commonly interrogated in studies of ferroptosis sensitivity, drug response, and stress-induced transcriptional programs.

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

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

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