Pitx2 CRISPR Activation Plasmid (h): sc-401426-ACT

PITX2 encodes the human paired-like homeodomain transcription factor Pitx2, a key regulator of developmental patterning and organogenesis. Pitx2 integrates upstream WNT/β-catenin and TGF-β–related signaling inputs to control transcriptional programs that govern cell fate specification, left–right asymmetry, and differentiation in ocular, craniofacial, cardiac, and dental lineages. Dysregulated PITX2 expression or function is linked to congenital malformations and has been investigated in contexts of aberrant epithelial–mesenchymal regulation and tumor-associated transcriptional networks. As a nuclear DNA-binding protein, Pitx2 is frequently studied for its promoter/enhancer occupancy, cofactor interactions, and downstream gene circuits that shape lineage identity.

Pitx2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous PITX2 expression without altering the underlying DNA sequence.

Pitx2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the PITX2 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 PITX2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Pitx2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native PITX2 locus and enabling the study of Pitx2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Pitx2 pathway restoration in tumor cells with silenced or reduced PITX2 expression.

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