Occludin CRISPR Activation Plasmid (h): sc-418271-ACT

Human OCLN encodes occludin, an integral membrane component of tight junction strands that regulates paracellular barrier permeability and epithelial cell polarity. Occludin participates in junctional assembly and remodeling through interactions with ZO scaffold proteins and links to the actin cytoskeleton, integrating signals from Rho GTPases, PKC, and MAPK pathways. Its phosphorylation state influences tight junction stability, cell–cell adhesion, and responses to inflammatory cues. Dysregulated OCLN expression or localization is associated with barrier defects and contributes to research models of intestinal and airway inflammation, blood–brain barrier integrity, and epithelial-to-mesenchymal transition.

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

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

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