P2X6 CRISPR Activation Plasmid (h): sc-416264-ACT

Human P2RX6 encodes the P2X6 subunit of ATP-gated P2X ion channels, which mediate rapid cation influx in response to extracellular nucleotides. P2X6 can contribute to heteromeric channel assemblies that shape Ca2+-dependent signaling, membrane excitability, and downstream transcriptional programs linked to purinergic neurotransmission and immune modulation. Through purinergic signaling networks, P2X6-associated activity can influence processes such as calcium homeostasis, vesicular release, and inflammatory cue integration. Dysregulated extracellular ATP sensing and ionotropic purinergic signaling have been implicated in neuroinflammatory and cardiovascular pathophysiology, motivating mechanistic studies of P2RX6 regulation in relevant cell types.

P2X6 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous P2RX6 expression without altering the underlying DNA sequence.

P2X6 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the P2RX6 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 P2RX6 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous P2X6 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native P2RX6 locus and enabling the study of P2X6-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of P2X6 pathway restoration in tumor cells with silenced or reduced P2RX6 expression.

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