IDO CRISPR Activation Plasmid (m): sc-421024-ACT

Mouse Ido1 encodes indoleamine 2,3-dioxygenase (IDO), a heme-dependent enzyme that catalyzes the first and rate-limiting step of tryptophan catabolism along the kynurenine pathway. By regulating local tryptophan availability and generating bioactive kynurenine metabolites, IDO influences immune cell activation, antigen presentation, and inflammatory signaling programs, including pathways linked to aryl hydrocarbon receptor (AHR) activity and cellular stress responses. Ido1 expression is commonly induced by interferon-γ and other inflammatory cues, connecting it to mechanisms of immune tolerance and tissue homeostasis. Dysregulated IDO activity has been associated with tumor immune evasion, chronic infection-associated inflammation, autoimmunity-related immune regulation, and neuroinflammatory processes, making it a useful node for immunometabolism research.

IDO CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Ido1 expression without altering the underlying DNA sequence.

IDO CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Ido1 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 Ido1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous IDO expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Ido1 locus and enabling the study of IDO-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of IDO pathway restoration in tumor cells with silenced or reduced Ido1 expression.

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