BNIP-3 CRISPR Activation Plasmid (h): sc-400985-ACT

BNIP3 encodes BNIP-3, a hypoxia-inducible BH3-only protein that localizes to mitochondria and regulates mitochondrial quality control, mitophagy, and cell death decisions. Through interactions with BCL2 family members and autophagy machinery, BNIP-3 links oxygen sensing to mitochondrial membrane remodeling, metabolic adaptation, and stress-responsive apoptosis or autophagic clearance. BNIP-3 is transcriptionally controlled by hypoxia signaling pathways such as HIF-1 and participates in cellular responses to ischemia, nutrient deprivation, and oxidative stress. Dysregulated BNIP3 expression has been associated with altered tumor hypoxia biology, cardiomyocyte stress responses, and neurodegenerative processes where mitochondrial dysfunction and impaired mitophagy contribute to pathology.

BNIP-3 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous BNIP3 expression without altering the underlying DNA sequence.

BNIP-3 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the BNIP3 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 BNIP3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous BNIP-3 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native BNIP3 locus and enabling the study of BNIP-3-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of BNIP-3 pathway restoration in tumor cells with silenced or reduced BNIP3 expression.

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