TRPV4 CRISPR Activation Plasmid (h): sc-401432-ACT

TRPV4 encodes a Ca2+-permeable, mechanosensitive TRP ion channel that integrates osmotic, thermal, and mechanical stimuli to regulate membrane excitability and intracellular calcium signaling. TRPV4 activity couples to downstream pathways including calmodulin-dependent signaling, MAPK cascades, and cytoskeletal remodeling, influencing processes such as epithelial barrier function, endothelial shear responses, and chondrocyte mechanotransduction. In human tissues, TRPV4 contributes to sensory transduction and volume homeostasis across diverse cell types. Altered TRPV4 signaling has been associated with pathophysiology spanning neuropathic pain mechanisms, skeletal dysplasias, and pulmonary and vascular dysfunction, making it a useful target for studying stimulus-dependent Ca2+ signaling networks.

TRPV4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous TRPV4 expression without altering the underlying DNA sequence.

TRPV4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the TRPV4 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 TRPV4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous TRPV4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native TRPV4 locus and enabling the study of TRPV4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of TRPV4 pathway restoration in tumor cells with silenced or reduced TRPV4 expression.

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