MARCH6 CRISPR Activation Plasmid (h): sc-437273-ACT

Human MARCH6 encodes a membrane-associated RING-CH E3 ubiquitin ligase localized primarily to the endoplasmic reticulum, where it coordinates ubiquitin-dependent turnover of misfolded or regulated membrane proteins. It participates in ER-associated degradation and broader proteostasis programs by coupling substrate recognition to ubiquitination and proteasomal clearance, thereby influencing lipid and sterol homeostasis pathways. Through modulation of protein quality control and metabolic signaling, altered MARCH6 activity can reshape cellular stress responses and downstream transcriptional programs relevant to dysregulated growth and inflammation. These functions make MARCH6 a useful target for mechanistic studies linking ubiquitin signaling, ER stress, and metabolic remodeling in disease-relevant cellular models.

MARCH6 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous MARCH6 expression without altering the underlying DNA sequence.

MARCH6 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the MARCH6 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 MARCH6 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous MARCH6 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native MARCH6 locus and enabling the study of MARCH6-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of MARCH6 pathway restoration in tumor cells with silenced or reduced MARCH6 expression.

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