MIB1 CRISPR Activation Plasmid (h): sc-404769-ACT

Human MIB1 encodes mindbomb E3 ubiquitin protein ligase 1, an E3 ubiquitin ligase that promotes ubiquitination and endocytosis of Notch ligands such as JAG1 and DLL proteins, thereby enabling productive Notch receptor activation. Through regulation of ligand trafficking and receptor–ligand signaling competence, MIB1 influences cell fate decisions, differentiation, and tissue patterning across developmental and homeostatic contexts. MIB1 activity interfaces with ubiquitin-dependent proteostasis and endocytic sorting pathways, shaping transcriptional outputs downstream of Notch signaling. Dysregulated MIB1/Notch axis function has been associated with altered neurogenesis, cardiovascular development phenotypes, and oncogenic signaling states, motivating mechanistic studies in diverse cellular models.

MIB1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous MIB1 expression without altering the underlying DNA sequence.

MIB1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the MIB1 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 MIB1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous MIB1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native MIB1 locus and enabling the study of MIB1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of MIB1 pathway restoration in tumor cells with silenced or reduced MIB1 expression.

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