RNF4 CRISPR Activation Plasmid (m): sc-422689-ACT

Mouse Rnf4 encodes RNF4, a SUMO-targeted ubiquitin E3 ligase that recognizes poly-SUMOylated substrates through SUMO-interacting motifs and promotes their ubiquitination and proteasomal turnover. RNF4 helps couple SUMO signaling to ubiquitin-dependent protein quality control, influencing DNA damage responses, replication stress, and transcriptional regulation through modulation of key nuclear factors. By controlling the stability of SUMO-modified proteins, RNF4 contributes to maintenance of genome integrity and cell-cycle progression, processes frequently perturbed in oncogenic transformation. Rnf4 perturbation is therefore used to study SUMO–ubiquitin pathway crosstalk, nuclear proteostasis, and stress-adaptive signaling networks in mammalian cells.

RNF4 CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Rnf4 expression without altering the underlying DNA sequence.

RNF4 CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Rnf4 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 Rnf4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous RNF4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Rnf4 locus and enabling the study of RNF4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of RNF4 pathway restoration in tumor cells with silenced or reduced Rnf4 expression.

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