ephrin-B3 CRISPR Activation Plasmid (h): sc-402767-ACT

EFNB3 encodes ephrin-B3, a transmembrane ligand for Eph receptor tyrosine kinases that mediates contact-dependent signaling in neural and vascular tissues. Ephrin-B3–Eph interactions regulate axon guidance, synaptic organization, cell migration, and boundary formation through bidirectional signaling that engages Rho family GTPases, MAPK cascades, and cytoskeletal remodeling pathways. In the nervous system, ephrin-B3 contributes to neurite outgrowth control and midline repulsion processes important for circuit assembly and plasticity. Dysregulated Eph/ephrin signaling, including altered EFNB3 expression, has been associated with aberrant cell motility and invasive phenotypes across multiple disease-relevant contexts, supporting its use as a mechanistic node in pathway studies.

ephrin-B3 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous EFNB3 expression without altering the underlying DNA sequence.

ephrin-B3 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the EFNB3 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 EFNB3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous ephrin-B3 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native EFNB3 locus and enabling the study of ephrin-B3-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of ephrin-B3 pathway restoration in tumor cells with silenced or reduced EFNB3 expression.

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