NKCC1 CRISPR Activation Plasmid (m): sc-422971-ACT

Mouse Slc12a2 encodes NKCC1 (SLC12A2), an electroneutral Na⁺-K⁺-2Cl⁻ cotransporter that regulates intracellular chloride homeostasis, cell volume control, and epithelial fluid secretion. By setting cytosolic Cl⁻ levels, NKCC1 influences membrane excitability and inhibitory neurotransmission through interactions with GABAergic signaling and broader ion-transport networks. NKCC1 activity contributes to osmotic stress responses and transepithelial salt movement in secretory epithelia, linking it to physiological processes in the nervous system, lung, and gastrointestinal tract. Dysregulated NKCC1-dependent chloride transport has been associated with models of neurological excitability and inflammatory airway and intestinal phenotypes, supporting mechanistic studies of ion balance in disease-relevant contexts.

NKCC1 CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Slc12a2 expression without altering the underlying DNA sequence.

NKCC1 CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Slc12a2 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 Slc12a2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous NKCC1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Slc12a2 locus and enabling the study of NKCC1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of NKCC1 pathway restoration in tumor cells with silenced or reduced Slc12a2 expression.

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