THP CRISPR Activation Plasmid (h): sc-401238-ACT

UMOD encodes uromodulin (THP), the most abundant glycoprotein secreted into normal human urine and a hallmark product of thick ascending limb epithelial cells. THP is synthesized in the endoplasmic reticulum, undergoes extensive glycosylation and GPI-anchoring, and is released apically to influence tubular homeostasis, protein aggregation, and interactions with urinary ions and microbes. UMOD expression and protein maturation are linked to epithelial proteostasis pathways, including ER quality control and trafficking through the Golgi, and can modulate inflammatory signaling within the renal tubular microenvironment. Genetic variation or dysregulated UMOD expression is associated with kidney disease susceptibility and tubulointerstitial pathology, making it a useful locus for studying renal epithelial function and stress responses.

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

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

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