Bag-3 CRISPR Activation Plasmid (h): sc-402929-ACT

BAG3 (Bag-3) is a stress-inducible co-chaperone that binds HSP70/HSC70 to coordinate protein quality control, balancing proteostasis, autophagy, and apoptosis in human cells. It supports chaperone-assisted selective autophagy and influences cytoskeletal organization, cell adhesion, and mechanotransduction, particularly under proteotoxic or oxidative stress. Through these functions, BAG3 contributes to regulation of cell survival pathways and turnover of damaged or aggregation-prone proteins. Dysregulated BAG3 expression or function has been linked to cardiomyopathy and neuromuscular disorders and is frequently studied in the context of tumor cell stress adaptation and resistance to proteotoxic stress.

Bag-3 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous BAG3 expression without altering the underlying DNA sequence.

Bag-3 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the BAG3 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 BAG3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Bag-3 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native BAG3 locus and enabling the study of Bag-3-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Bag-3 pathway restoration in tumor cells with silenced or reduced BAG3 expression.

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