Ox40 CRISPR Activation Plasmid (h): sc-401326-ACT

Human TNFRSF4 encodes OX40 (CD134), an inducible TNF receptor superfamily costimulatory molecule primarily expressed on activated T cells that reinforces antigen-driven responses and supports effector and memory differentiation. OX40 ligation promotes recruitment of TRAF adaptor proteins and activation of NF-κB, MAPK, and PI3K–AKT signaling, shaping cytokine production, survival, and proliferation in adaptive immunity. Dysregulated OX40–OX40L signaling has been associated with altered immune tolerance and inflammatory programs observed across autoimmunity, transplantation biology, and tumor immune microenvironments. TNFRSF4 is therefore widely studied in T cell activation, costimulation checkpoints, and immune cell–cell interaction models.

Ox40 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous TNFRSF4 expression without altering the underlying DNA sequence.

Ox40 CRISPR Activation Plasmid (h) 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.