Alix CRISPR Activation Plasmid (h): sc-400350-ACT

PDCD6IP encodes Alix, an ESCRT-associated adaptor that coordinates endosomal sorting, multivesicular body biogenesis, cytokinetic abscission, and plasma membrane repair through interactions with ESCRT-III components and syntenin. Alix also participates in exosome formation and receptor downregulation, influencing signaling pathways that depend on membrane trafficking and ubiquitin-dependent cargo selection. Through its roles in membrane remodeling and cytoskeletal coordination, Alix impacts autophagy-related processes and viral budding of enveloped viruses that hijack ESCRT machinery. Dysregulation of PDCD6IP/Alix-linked trafficking and extracellular vesicle dynamics has been associated with phenotypes relevant to cancer cell invasion, neurodegeneration, and altered immune signaling, making it a useful node for mechanistic pathway studies.

Alix CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous PDCD6IP expression without altering the underlying DNA sequence.

Alix CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the PDCD6IP 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 PDCD6IP transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Alix expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native PDCD6IP locus and enabling the study of Alix-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Alix pathway restoration in tumor cells with silenced or reduced PDCD6IP expression.

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