MDA5 CRISPR Activation Plasmid (m): sc-427977-ACT

Mouse Ifih1 encodes MDA5 (IFIH1), a cytosolic DExD/H-box RNA helicase that detects long double-stranded RNA produced during viral replication and other sources of aberrant RNA. Upon ligand engagement, MDA5 signals through the MAVS adaptor to activate IRF3/IRF7 and NF-κB pathways, inducing type I interferons and interferon-stimulated genes that shape innate and adaptive immune responses. This RNA-sensing axis influences antiviral restriction, inflammatory cytokine programs, and cross-talk with autophagy and stress granule dynamics. Dysregulated MDA5 activity and interferon signaling have been linked to interferonopathies and autoimmune-like inflammation, and are also relevant to tumor immune sensing and host–pathogen interactions in experimental models.

MDA5 CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Ifih1 expression without altering the underlying DNA sequence.

MDA5 CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Ifih1 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 Ifih1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous MDA5 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Ifih1 locus and enabling the study of MDA5-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of MDA5 pathway restoration in tumor cells with silenced or reduced Ifih1 expression.

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