ILT-2 CRISPR Activation Plasmid (h): sc-403335-ACT

Human LILRB1 encodes ILT-2 (LIR-1/CD85j), an inhibitory immunoreceptor expressed on NK cells, monocytes, dendritic cells, and subsets of T and B cells that binds classical and nonclassical HLA class I molecules, including HLA-G. Upon ligand engagement, ILT-2 signals through ITIM motifs to recruit SHP-1/SHP-2 phosphatases, dampening activating receptor pathways and reducing cytotoxicity, cytokine production, and antigen-presenting cell maturation. This checkpoint-like signaling shapes immune tolerance at tissue interfaces and modulates responses to infection and cellular stress. Dysregulated LILRB1/ILT-2 activity has been implicated in altered immune regulation across inflammatory and autoimmune contexts and in immune evasion mechanisms within disease microenvironments.

ILT-2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous LILRB1 expression without altering the underlying DNA sequence.

ILT-2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the LILRB1 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 LILRB1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous ILT-2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native LILRB1 locus and enabling the study of ILT-2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of ILT-2 pathway restoration in tumor cells with silenced or reduced LILRB1 expression.

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