IAP CRISPR Activation Plasmid (h): sc-401544-ACT

Human ALPI encodes intestinal alkaline phosphatase (IAP), a glycosylphosphatidylinositol-anchored brush-border enzyme that hydrolyzes phosphate monoesters and dephosphorylates microbial-associated ligands in the gut lumen. Through modulation of luminal nucleotide pools and detoxification of pro-inflammatory phosphorylated molecules, IAP contributes to epithelial barrier homeostasis, mucosal innate immune signaling, and microbial ecology. ALPI activity is linked to processes in intestinal differentiation and enterocyte function, intersecting with pathways that shape inflammatory tone and nutrient handling at the mucosal surface. Dysregulated ALPI expression or activity has been associated with intestinal inflammatory phenotypes and altered host–microbiome interactions relevant to gastrointestinal disease models.

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

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

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