PBGD CRISPR Activation Plasmid (m): sc-420870-ACT

Mouse Hmbs encodes porphobilinogen deaminase (PBGD), a cytosolic enzyme that catalyzes the polymerization of four porphobilinogen molecules to form hydroxymethylbilane, a central intermediate in the heme biosynthesis pathway. By supporting heme availability, PBGD influences mitochondrial respiration, cytochrome function, and redox homeostasis, with downstream effects on erythroid maturation and cellular oxidative metabolism. Perturbation of HMBS/PBGD activity is linked to disordered porphyrin metabolism and accumulation of upstream intermediates, making this gene a key node for modeling metabolic stress responses. Hmbs is therefore relevant to studies of hepatic and hematopoietic physiology, mitochondrial bioenergetics, and pathway-level regulation of tetrapyrrole synthesis.

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

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

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