Npl4 CRISPR Activation Plasmid (h): sc-403787-ACT

Human NPLOC4 encodes Npl4, a core cofactor of the p97/VCP ATPase that functions with UFD1 to recognize and extract ubiquitinated substrates for processing by the proteasome. Through its ubiquitin-binding domains, Npl4 helps coordinate ER-associated degradation (ERAD), clearance of stalled ribosome-associated nascent chains, and broader proteostasis control that influences cell-cycle progression and stress-adaptive signaling. These pathways intersect with DNA damage responses and inflammatory transcriptional programs by regulating the turnover of key signaling and chromatin-associated proteins. Dysregulation of the VCP–UFD1–NPL4 axis is linked to proteotoxic stress, altered protein homeostasis, and vulnerability in proliferative states, making NPLOC4 a relevant target for mechanistic studies in neurodegeneration and cancer biology.

Npl4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous NPLOC4 expression without altering the underlying DNA sequence.

Npl4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the NPLOC4 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 NPLOC4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Npl4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native NPLOC4 locus and enabling the study of Npl4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Npl4 pathway restoration in tumor cells with silenced or reduced NPLOC4 expression.

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