OPG/Osteoprotegerin CRISPR Activation Plasmid (h): sc-400497-ACT

TNFRSF11B encodes osteoprotegerin (OPG), a secreted decoy receptor in the TNF receptor superfamily that binds RANKL (TNFSF11) and limits RANK–RANKL signaling, thereby modulating osteoclast differentiation and bone remodeling. By sequestering RANKL and related ligands, OPG influences downstream NF-κB and MAPK pathway activation in osteoclast precursors and contributes to coupling between osteoblast and osteoclast activity. Beyond skeletal biology, OPG has been implicated in vascular and immune-associated processes through regulation of cytokine signaling and extracellular microenvironment cues. Dysregulated TNFRSF11B/OPG expression is associated with altered bone turnover phenotypes and has been studied in contexts including osteoporosis, osteolytic disease mechanisms, and calcification-related pathobiology.

OPG/Osteoprotegerin CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous TNFRSF11B expression without altering the underlying DNA sequence.

OPG/Osteoprotegerin CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the TNFRSF11B 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 TNFRSF11B transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous OPG/Osteoprotegerin expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native TNFRSF11B locus and enabling the study of OPG/Osteoprotegerin-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of OPG/Osteoprotegerin pathway restoration in tumor cells with silenced or reduced TNFRSF11B expression.

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