IRS-4 CRISPR Activation Plasmid (h): sc-402033-ACT

Human IRS4 encodes insulin receptor substrate 4 (IRS-4), a cytoplasmic adaptor that becomes tyrosine phosphorylated downstream of insulin and IGF-1 receptors to couple receptor activation to PI3K–AKT–mTOR and RAS–MAPK signaling. By serving as a docking platform for SH2-domain proteins such as PI3K regulatory subunits and GRB2-associated factors, IRS-4 influences glucose and lipid metabolism programs, cell growth, and survival decisions. IRS4 expression and signaling rewiring have been linked to altered growth factor dependence and proliferative phenotypes in cancer-related contexts, making it a useful node for interrogating pathway crosstalk and feedback regulation in receptor tyrosine kinase networks.

IRS-4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous IRS4 expression without altering the underlying DNA sequence.

IRS-4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the IRS4 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 IRS4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous IRS-4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native IRS4 locus and enabling the study of IRS-4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of IRS-4 pathway restoration in tumor cells with silenced or reduced IRS4 expression.

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