RANK CRISPR Activation Plasmid (m): sc-423439-ACT

Mouse Tnfrsf11a encodes RANK, a TNF receptor superfamily member that functions as a central signal transducer for RANKL-dependent communication between immune, bone, and stromal compartments. RANK activation engages TRAF adaptors to stimulate NF-κB, MAPK (ERK/JNK/p38), and PI3K–AKT signaling, coordinating osteoclast differentiation, survival, and inflammatory gene expression programs. In vivo and in vitro, altered RANK pathway activity is closely linked to dysregulated bone remodeling and immune microenvironment crosstalk, making Tnfrsf11a a key node for studying osteoimmunology and tissue homeostasis. As a receptor with context-dependent outputs, RANK provides a tractable entry point to interrogate downstream transcriptional networks and ligand-driven cellular phenotypes.

RANK CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Tnfrsf11a expression without altering the underlying DNA sequence.

RANK CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Tnfrsf11a 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 Tnfrsf11a transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous RANK expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Tnfrsf11a locus and enabling the study of RANK-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of RANK pathway restoration in tumor cells with silenced or reduced Tnfrsf11a expression.

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