CD28 CRISPR Activation Plasmid (h): sc-401525-ACT

Human CD28 encodes a costimulatory receptor expressed on T cells that binds B7 family ligands (CD80/CD86) to amplify T cell receptor signaling and support full activation. CD28 engagement promotes IL-2 production, survival, and metabolic reprogramming through PI3K–AKT–mTOR, NF-κB, and MAPK pathways, influencing proliferation, differentiation, and cytokine output. Dysregulated CD28 signaling is implicated in immune-mediated pathology and tumor immune evasion, and altered expression or signaling can affect responses in autoimmunity, chronic infection, and cancer immunology models. CD28 is therefore a key node for studying T cell activation thresholds, checkpoint interactions, and immune microenvironment dynamics.

CD28 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CD28 expression without altering the underlying DNA sequence.

CD28 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CD28 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 CD28 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous CD28 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CD28 locus and enabling the study of CD28-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of CD28 pathway restoration in tumor cells with silenced or reduced CD28 expression.

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