CD223 CRISPR Activation Plasmid (m): sc-421371-ACT

Lag3 encodes CD223, an inhibitory immune checkpoint receptor expressed on activated T cells, regulatory T cells, NK cells, and subsets of dendritic cells in mouse. CD223 engagement modulates TCR signaling strength and shapes effector differentiation by attenuating proximal activation pathways, contributing to peripheral tolerance and control of inflammatory responses. In immune microenvironments with persistent antigen exposure, CD223 is linked to transcriptional programs associated with T cell dysfunction and altered cytokine output. Because of its role in checkpoint signaling and immune homeostasis, Lag3 is frequently studied in models of chronic inflammation, infection, and tumor immunology to dissect mechanisms of immune regulation.

CD223 CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Lag3 expression without altering the underlying DNA sequence.

CD223 CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Lag3 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 Lag3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous CD223 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Lag3 locus and enabling the study of CD223-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of CD223 pathway restoration in tumor cells with silenced or reduced Lag3 expression.

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