RNF181 CRISPR Activation Plasmid (h): sc-403952-ACT

RNF181 encodes a RING finger E3 ubiquitin ligase that promotes substrate ubiquitination to regulate protein turnover and signaling output. Through ubiquitin-dependent modulation of key signaling intermediates, RNF181 is implicated in control of immune and inflammatory responses, cellular stress adaptation, and maintenance of protein homeostasis. Altered RNF181 expression or activity can perturb pathway amplitude and timing, linking its regulation to mechanisms relevant to oncogenic signaling, immune dysfunction, and other conditions where ubiquitin signaling is dysregulated. These features make RNF181 a useful node for dissecting ubiquitin-mediated pathway control and transcriptional state changes in human cell models.

RNF181 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RNF181 expression without altering the underlying DNA sequence.

RNF181 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RNF181 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 RNF181 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous RNF181 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RNF181 locus and enabling the study of RNF181-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of RNF181 pathway restoration in tumor cells with silenced or reduced RNF181 expression.

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