Ox40L CRISPR Activation Plasmid (h): sc-403518-ACT

TNFSF4 encodes OX40L (CD252), a TNF superfamily ligand predominantly expressed on antigen-presenting cells that engages the OX40 (TNFRSF4) receptor on activated T cells to promote costimulation, proliferation, and survival. OX40L–OX40 signaling amplifies downstream NF-κB, MAPK, and PI3K/AKT pathway activity, shaping effector differentiation, memory formation, and cytokine programs in adaptive immunity. Dysregulated TNFSF4 expression has been linked to inflammatory immune microenvironments and altered tolerance mechanisms, with relevance to autoimmune-associated genetic risk and tumor immunology research. As a membrane-bound ligand, OX40L also contributes to cell–cell communication within lymphoid tissues and inflamed sites, influencing B cell help and antigen-driven responses.

Ox40L CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous TNFSF4 expression without altering the underlying DNA sequence.

Ox40L CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the TNFSF4 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 TNFSF4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Ox40L expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native TNFSF4 locus and enabling the study of Ox40L-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Ox40L pathway restoration in tumor cells with silenced or reduced TNFSF4 expression.

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