NKCC2 CRISPR Activation Plasmid (h): sc-401707-ACT

Human SLC12A1 encodes NKCC2 (Sodium/potassium/chloride cotransporter 2), an apical membrane transporter that mediates electroneutral Na⁺-K⁺-2Cl⁻ uptake in epithelial cells, most prominently in the thick ascending limb of the nephron. By coupling chloride transport to sodium and potassium flux, NKCC2 supports transepithelial salt reabsorption and contributes to osmotic balance and tubular electrolyte handling. NKCC2 activity interfaces with ion homeostasis programs regulated by cAMP/PKA signaling, WNK–SPAK/OSR1 kinase pathways, and membrane trafficking processes that tune transporter abundance at the cell surface. Altered SLC12A1/NKCC2 function is implicated in inherited salt-wasting tubulopathies and is widely studied in the context of renal physiology, electrolyte disorders, and mechanisms of diuretic response.

NKCC2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SLC12A1 expression without altering the underlying DNA sequence.

NKCC2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SLC12A1 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 SLC12A1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous NKCC2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SLC12A1 locus and enabling the study of NKCC2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of NKCC2 pathway restoration in tumor cells with silenced or reduced SLC12A1 expression.

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