TRC8 CRISPR Activation Plasmid (m): sc-429220-ACT

Rnf139 encodes TRC8, an endoplasmic reticulum–resident RING finger E3 ubiquitin ligase implicated in ubiquitin-dependent protein turnover and ER-associated degradation. Through coupling ubiquitination to proteasomal clearance, TRC8 contributes to proteostasis and can influence lipid and sterol-regulated processes via interactions with membrane-bound signaling and metabolic regulators. Altered TRC8 activity has been linked to dysregulated growth control and cellular stress responses, making Rnf139 a useful node for studying ubiquitin signaling in pathways relevant to genome stability, metabolic homeostasis, and oncogenic transformation. In mouse models, modulation of Rnf139 expression supports mechanistic studies of tissue-specific proteostasis and signaling rewiring.

TRC8 CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Rnf139 expression without altering the underlying DNA sequence.

TRC8 CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Rnf139 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 Rnf139 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous TRC8 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Rnf139 locus and enabling the study of TRC8-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of TRC8 pathway restoration in tumor cells with silenced or reduced Rnf139 expression.

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