Calnexin CRISPR Activation Plasmid (m2): sc-419436-ACT-2

Mouse Canx encodes calnexin, an endoplasmic reticulum (ER) membrane chaperone that binds monoglucosylated N-linked glycans to promote folding, assembly, and retention of nascent glycoproteins within the calnexin/calreticulin cycle. Calnexin functions in ER quality control and proteostasis by coordinating with ERp57 and related oxidoreductases, influencing disulfide bond formation, ER-associated degradation (ERAD), and the unfolded protein response during ER stress. Disruption of Canx-dependent folding surveillance can perturb secretory pathway homeostasis and has been linked to phenotypes relevant to neurodevelopment, myelination, and immune function in mouse models where glycoprotein maturation is critical. Gene editing of Canx supports mechanistic studies of glycoprotein biogenesis, ER stress signaling, and trafficking of membrane and secreted proteins across cell types and disease-relevant contexts.

Calnexin CRISPR Activation Plasmid (m2) provides a targeted, non-destructive approach to upregulating endogenous Canx expression without altering the underlying DNA sequence.

Calnexin CRISPR Activation Plasmid (m2) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Canx 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 Canx transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Calnexin expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Canx locus and enabling the study of Calnexin-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Calnexin pathway restoration in tumor cells with silenced or reduced Canx expression.

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