NCCT CRISPR Activation Plasmid (h): sc-402965-ACT

SLC12A3 encodes NCCT (NCC), an electroneutral Na⁺/Cl⁻ cotransporter localized to the apical membrane of distal convoluted tubule epithelial cells where it mediates sodium and chloride reabsorption and contributes to electrolyte homeostasis and blood pressure regulation. NCCT activity is integrated with renal salt-handling networks involving WNK–SPAK/OSR1 kinase signaling, which modulates transporter phosphorylation, trafficking, and membrane abundance. Genetic variation or altered expression of SLC12A3 is linked to inherited salt-wasting tubulopathies such as Gitelman syndrome and has been investigated in the context of hypertension susceptibility and renal electrolyte phenotypes. As a kidney-enriched transporter, NCCT provides a tractable node for studying epithelial ion transport, cell polarity, and osmoregulatory signaling in human model systems.

NCCT CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SLC12A3 expression without altering the underlying DNA sequence.

NCCT CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SLC12A3 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 SLC12A3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous NCCT expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SLC12A3 locus and enabling the study of NCCT-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of NCCT pathway restoration in tumor cells with silenced or reduced SLC12A3 expression.

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