connexin 26 CRISPR Activation Plasmid (h): sc-402132-ACT

GJB2 encodes connexin 26, a four-pass transmembrane gap junction protein that assembles into hexameric hemichannels and intercellular channels to mediate direct cell–cell transfer of ions and small metabolites. Connexin 26 supports epithelial and sensory tissue homeostasis by coordinating electrical coupling, calcium signaling, and metabolic synchronization, with additional roles in ATP release and paracrine communication via hemichannels under specific physiological contexts. This protein is central to gap junction communication networks that influence differentiation, barrier function, and stress responses. Genetic variation in GJB2 is strongly associated with hereditary hearing loss and is also linked to syndromic disorders affecting skin and adnexal tissues, making it a widely studied gene in auditory biology and epithelial physiology.

connexin 26 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous GJB2 expression without altering the underlying DNA sequence.

connexin 26 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the GJB2 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 GJB2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous connexin 26 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native GJB2 locus and enabling the study of connexin 26-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of connexin 26 pathway restoration in tumor cells with silenced or reduced GJB2 expression.

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