GLI-3 CRISPR Activation Plasmid (h): sc-400853-ACT

GLI3 encodes the GLI-3 zinc-finger transcription factor, a central effector and context-dependent repressor/activator within the Hedgehog (HH) signaling pathway. Through regulated proteolytic processing and nuclear transcriptional control, GLI-3 integrates cues from primary cilia and modulates developmental programs governing patterning, stem/progenitor cell behavior, and tissue morphogenesis. Dysregulated GLI3 activity perturbs HH target gene expression networks and has been linked to congenital malformation syndromes and pathway-driven oncogenic phenotypes where HH–GLI transcriptional output is altered. As a transcriptional node downstream of PTCH1/SMO, GLI-3 is broadly used to interrogate HH pathway dynamics, ciliary signaling, and transcriptional regulation in human cell models.

GLI-3 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous GLI3 expression without altering the underlying DNA sequence.

GLI-3 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the GLI3 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 GLI3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous GLI-3 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native GLI3 locus and enabling the study of GLI-3-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of GLI-3 pathway restoration in tumor cells with silenced or reduced GLI3 expression.

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