patched 2 CRISPR Activation Plasmid (h): sc-404866-ACT

PTCH2 encodes patched 2, a twelve-pass transmembrane receptor that binds Hedgehog ligands and functions as a key negative regulator of Smoothened (SMO) signaling. By modulating GLI transcription factor activity, PTCH2 contributes to control of cell fate specification, proliferation, and tissue patterning during development, with continued relevance to epithelial homeostasis in adult tissues. Dysregulated Hedgehog pathway signaling, including altered PTCH2 expression or function, has been associated with aberrant growth control and tumor biology in several tissue contexts, making PTCH2 a useful node for mechanistic studies of pathway rewiring. PTCH2 also provides a tractable target for investigating crosstalk between Hedgehog signaling and other regulatory programs that influence differentiation and cell-cycle progression.

patched 2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous PTCH2 expression without altering the underlying DNA sequence.

patched 2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the PTCH2 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 PTCH2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous patched 2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native PTCH2 locus and enabling the study of patched 2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of patched 2 pathway restoration in tumor cells with silenced or reduced PTCH2 expression.

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