CD3-ε CRISPR Activation Plasmid (h): sc-400240-ACT

CD3E encodes CD3-ε, an essential component of the T cell receptor (TCR)-CD3 complex that couples antigen recognition to intracellular signaling required for thymocyte development and peripheral T cell activation. Through its immunoreceptor tyrosine-based activation motifs (ITAMs) within the CD3 chains, CD3-ε supports proximal kinase cascades involving LCK and ZAP70 and propagates downstream pathways such as NF-κB, NFAT, and MAPK to regulate cytokine production, proliferation, and differentiation. Perturbation of CD3E expression or CD3 complex assembly can disrupt TCR signaling thresholds, alter immune homeostasis, and contribute to immune dysregulation phenotypes. As a lineage-defining molecule in T cells, CD3-ε is widely used in studies of receptor signaling architecture, T cell functional states, and immune-oncology models in vitro.

CD3-ε CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CD3E expression without altering the underlying DNA sequence.

CD3-ε CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CD3E 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 CD3E transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous CD3-ε expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CD3E locus and enabling the study of CD3-ε-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of CD3-ε pathway restoration in tumor cells with silenced or reduced CD3E expression.

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