UPII CRISPR Activation Plasmid (h): sc-402242-ACT

UPK2 encodes uroplakin II (UPII), an integral component of the urothelial apical plaques that help form the asymmetric unit membrane and support the barrier properties of differentiated umbrella cells. UPII assembles with other uroplakins in the secretory pathway and is trafficked to the apical surface, contributing to membrane organization, epithelial polarity, and mechanical stability in the urinary tract. As a lineage-restricted marker of urothelial differentiation, altered UPK2 expression is widely used to study changes in epithelial maturation programs and cellular identity in bladder biology. Dysregulated uroplakin expression patterns are frequently investigated in the context of urothelial pathology, including tumor-associated differentiation states and altered barrier function.

UPII CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous UPK2 expression without altering the underlying DNA sequence.

UPII CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the UPK2 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 UPK2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous UPII expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native UPK2 locus and enabling the study of UPII-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of UPII pathway restoration in tumor cells with silenced or reduced UPK2 expression.

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