EphB4 CRISPR Activation Plasmid (m): sc-420201-ACT

Mouse Ephb4 encodes the EphB4 receptor tyrosine kinase, a member of the ephrin–Eph signaling system that mediates contact-dependent communication between adjacent cells. EphB4–ephrin-B2 interactions regulate vascular development and remodeling, including arterial–venous specification, endothelial cell migration, and guidance cues during tissue patterning. Downstream signaling interfaces with cytoskeletal dynamics and adhesion pathways, engaging Rho family GTPases, PI3K/AKT, MAPK/ERK, and focal adhesion signaling to influence cell positioning and boundary formation. Dysregulated EphB4 signaling has been implicated in pathological angiogenesis and tumor-associated vascular phenotypes, making it relevant for mechanistic studies in cancer biology, developmental models, and microenvironment research.

EphB4 CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Ephb4 expression without altering the underlying DNA sequence.

EphB4 CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Ephb4 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 Ephb4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous EphB4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Ephb4 locus and enabling the study of EphB4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of EphB4 pathway restoration in tumor cells with silenced or reduced Ephb4 expression.

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