Notch 3 CRISPR Activation Plasmid (h): sc-400424-ACT

NOTCH3 encodes Notch 3, a single-pass transmembrane receptor that mediates juxtacrine Notch signaling in response to Delta-like and Jagged ligands. Upon ligand engagement, proteolytic processing releases the Notch intracellular domain, which translocates to the nucleus and cooperates with RBPJ/CSL and co-activators to regulate transcriptional programs controlling cell fate specification, differentiation, and tissue homeostasis. Notch 3 activity is closely linked to vascular smooth muscle cell biology and broader developmental signaling networks, and dysregulation of NOTCH3-dependent transcription has been associated with inherited arteriopathies and oncogenic contexts. As a pathway node, NOTCH3 provides a tractable model for dissecting receptor activation dynamics, transcriptional output, and crosstalk with MAPK, PI3K-AKT, and TGF-β–related processes.

Notch 3 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous NOTCH3 expression without altering the underlying DNA sequence.

Notch 3 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the NOTCH3 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 NOTCH3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Notch 3 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native NOTCH3 locus and enabling the study of Notch 3-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Notch 3 pathway restoration in tumor cells with silenced or reduced NOTCH3 expression.

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