Pitx3 CRISPR Activation Plasmid (h): sc-402995-ACT

PITX3 encodes the paired-like homeodomain transcription factor Pitx3, a nuclear regulator that helps specify midbrain dopaminergic neuron identity and supports neuronal differentiation and maintenance. Pitx3 functions within developmental transcriptional networks that interface with Wnt/β-catenin signaling, retinoic acid–responsive programs, and other homeobox factors to shape lineage decisions and maturation states. Altered PITX3 regulation has been linked to dopaminergic system vulnerability and is studied in the context of neurodevelopmental and neurodegenerative disease biology, including pathways relevant to Parkinson’s disease. As a transcriptional controller, Pitx3 is also used to probe gene regulatory circuitry, chromatin state transitions, and cell-fate reprogramming in human cellular models.

Pitx3 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous PITX3 expression without altering the underlying DNA sequence.

Pitx3 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the PITX3 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 PITX3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Pitx3 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native PITX3 locus and enabling the study of Pitx3-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Pitx3 pathway restoration in tumor cells with silenced or reduced PITX3 expression.

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