Phox2b CRISPR Activation Plasmid (h): sc-401351-ACT

PHOX2B encodes the homeobox transcription factor Phox2b, a lineage-determining regulator required for specification and maintenance of autonomic and noradrenergic neuronal programs during development. Phox2b coordinates transcriptional networks governing neuronal differentiation, axon guidance, and catecholaminergic gene expression, including pathways associated with neural crest–derived cell fates and brainstem respiratory control circuits. Genetic alterations or dysregulated expression of PHOX2B are linked to neurocristopathies and pediatric neural crest tumors, including congenital central hypoventilation syndrome and subsets of neuroblastoma. These features make PHOX2B a key node for studying transcriptional control of autonomic nervous system development and disease-relevant neural differentiation states.

Phox2b CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous PHOX2B expression without altering the underlying DNA sequence.

Phox2b CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the PHOX2B 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 PHOX2B transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Phox2b expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native PHOX2B locus and enabling the study of Phox2b-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Phox2b pathway restoration in tumor cells with silenced or reduced PHOX2B expression.

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