GP78 CRISPR Activation Plasmid (h): sc-402346-ACT

AMFR encodes the human GP78 (autocrine motility factor receptor), an ER-anchored E3 ubiquitin ligase that functions as a central component of endoplasmic reticulum–associated degradation (ERAD) by ubiquitinating misfolded proteins for proteasomal clearance. GP78 cooperates with ER chaperones and E2 enzymes to regulate protein quality control, ER stress responses, and turnover of select metabolic and signaling regulators. Through its roles in ubiquitin-dependent proteostasis and membrane-associated signaling, AMFR/GP78 activity has been linked to cellular adaptation programs that influence proliferation, migration, and inflammatory stress signaling. Dysregulation of ERAD and ubiquitin ligase networks involving GP78 has been studied in contexts including cancer biology, neurodegeneration, and metabolic disease models where proteostasis imbalance is a contributing factor.

GP78 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous AMFR expression without altering the underlying DNA sequence.

GP78 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the AMFR 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 AMFR transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous GP78 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native AMFR locus and enabling the study of GP78-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of GP78 pathway restoration in tumor cells with silenced or reduced AMFR expression.

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