Ox40 CRISPR Activation Plasmid (m): sc-423560-ACT

Mouse Tnfrsf4 encodes OX40 (CD134), an inducible TNFR superfamily co-stimulatory receptor expressed primarily on activated T cells and subsets of regulatory T cells. OX40 engagement by OX40L promotes clonal expansion, survival, and effector differentiation through TRAF-dependent signaling that converges on NF-κB, MAPK, and PI3K–AKT pathways, supporting cytokine production and memory formation. Dysregulated OX40 signaling is implicated in inflammatory and autoimmune processes and shapes tumor immune contexture by altering T cell persistence and suppressive networks. As a result, Tnfrsf4 is widely studied in T cell activation thresholds, germinal center support, and immune microenvironment remodeling in mouse disease models.

Ox40 CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Tnfrsf4 expression without altering the underlying DNA sequence.

Ox40 CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Tnfrsf4 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 Tnfrsf4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Ox40 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Tnfrsf4 locus and enabling the study of Ox40-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Ox40 pathway restoration in tumor cells with silenced or reduced Tnfrsf4 expression.

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