RNF185 CRISPR Activation Plasmid (h): sc-416413-ACT

RNF185 encodes a RING finger E3 ubiquitin ligase primarily associated with the endoplasmic reticulum and mitochondria-associated membranes, where it helps regulate protein quality control and organelle homeostasis. By catalyzing ubiquitination of specific substrates, RNF185 influences proteostasis pathways including ER-associated degradation and stress-adaptive signaling, with downstream effects on mitochondrial dynamics. Reported roles in selective autophagy and innate immune regulation link RNF185 to mechanisms that coordinate cellular responses to damaged proteins and organelles. Dysregulated ubiquitin signaling and impaired proteostasis are recurrent features across neurodegeneration, metabolic disorders, and cancer biology, making RNF185 a useful node for pathway-focused research.

RNF185 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RNF185 expression without altering the underlying DNA sequence.

RNF185 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RNF185 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 RNF185 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous RNF185 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RNF185 locus and enabling the study of RNF185-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of RNF185 pathway restoration in tumor cells with silenced or reduced RNF185 expression.

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