AIP4 CRISPR Activation Plasmid (h): sc-401677-ACT

Human ITCH encodes the HECT-type E3 ubiquitin ligase AIP4, a key regulator of protein turnover that controls signal duration by catalyzing ubiquitination and proteasomal or lysosomal targeting of specific substrates. AIP4 modulates multiple pathways including T cell receptor signaling, NF-κB and MAPK signaling, and TGF-β/SMAD pathway dynamics, thereby influencing immune homeostasis, inflammation, and cellular stress responses. By regulating the stability and trafficking of receptors and signaling adaptors, ITCH impacts apoptosis, differentiation, and epithelial–mesenchymal transition-associated programs. Dysregulated ITCH activity or expression has been linked to immune dysregulation phenotypes and altered oncogenic signaling networks, making it a useful node for mechanistic studies of ubiquitin-dependent pathway control.

AIP4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ITCH expression without altering the underlying DNA sequence.

AIP4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ITCH 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 ITCH transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous AIP4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ITCH locus and enabling the study of AIP4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of AIP4 pathway restoration in tumor cells with silenced or reduced ITCH expression.

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