KCC3 CRISPR Activation Plasmid (h): sc-405975-ACT

SLC12A6 encodes the potassium–chloride cotransporter KCC3, a member of the cation-chloride cotransporter family that mediates electroneutral K+ and Cl− efflux to regulate intracellular chloride, osmotic balance, and cell volume. KCC3 activity contributes to maintenance of membrane potential and neuronal excitability by shaping chloride gradients that influence inhibitory neurotransmission, and it is modulated by phosphorylation-dependent signaling pathways responsive to cellular stress and ion homeostasis. In the nervous system, KCC3 supports axon and myelin integrity and participates in responses to swelling and ionic perturbation. Genetic disruption or dysregulation of SLC12A6 is associated with neurodevelopmental and neurodegenerative phenotypes, including peripheral neuropathy and white matter abnormalities, making it relevant for studying ion-transport mechanisms in disease-relevant cell types.

KCC3 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SLC12A6 expression without altering the underlying DNA sequence.

KCC3 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SLC12A6 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 SLC12A6 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous KCC3 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SLC12A6 locus and enabling the study of KCC3-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of KCC3 pathway restoration in tumor cells with silenced or reduced SLC12A6 expression.

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