TIM-3 CRISPR Activation Plasmid (h): sc-416365-ACT

HAVCR2 encodes T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), an immune checkpoint receptor expressed on activated T cells, regulatory T cells, NK cells, and myeloid subsets. TIM-3 modulates immune activation and exhaustion programs through ligand-dependent signaling with galectin-9, phosphatidylserine, CEACAM1, and HMGB1, influencing TCR signaling thresholds, cytokine production, and innate sensing. These processes intersect with inflammatory and tolerogenic pathways that shape anti-viral responses, tumor immune evasion, and myeloid-driven immunoregulation. Altered TIM-3 expression and signaling have been observed in contexts of chronic infection, autoimmunity, and cancer-associated immune dysfunction, supporting its use as a mechanistic marker for immune-state remodeling.

TIM-3 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous HAVCR2 expression without altering the underlying DNA sequence.

TIM-3 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the HAVCR2 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 HAVCR2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous TIM-3 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native HAVCR2 locus and enabling the study of TIM-3-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of TIM-3 pathway restoration in tumor cells with silenced or reduced HAVCR2 expression.

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