CD3-ζ CRISPR Activation Plasmid (h): sc-400591-ACT

CD247 encodes the CD3-zeta (CD3ζ) chain, an essential component of the T cell receptor (TCR)–CD3 complex that contains immunoreceptor tyrosine-based activation motifs (ITAMs) required for antigen-driven signaling. Upon TCR engagement, CD3ζ phosphorylation supports recruitment of ZAP70 and propagation through LAT/SLP-76 signalosomes to activate downstream MAPK, NF-κB, and NFAT pathways, shaping T cell activation, cytokine production, and cytotoxic effector function. Altered CD247 expression or signaling competence has been associated with immune dysregulation and impaired lymphocyte responses, including tumor-associated T cell dysfunction and autoimmune-related signaling imbalance. As a proximal adaptor in adaptive immunity, CD3ζ is frequently studied in T cell development, activation thresholds, and engineered receptor signaling.

CD3-ζ CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CD247 expression without altering the underlying DNA sequence.

CD3-ζ CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CD247 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 CD247 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous CD3-ζ expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CD247 locus and enabling the study of CD3-ζ-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of CD3-ζ pathway restoration in tumor cells with silenced or reduced CD247 expression.

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