p19 INK4D/CDKN2D CRISPR Activation Plasmid (h): sc-401761-ACT

CDKN2D encodes p19 INK4D, a member of the INK4 family of cyclin-dependent kinase inhibitors that selectively restrains CDK4 and CDK6 activity to enforce the G1/S checkpoint. By limiting phosphorylation of RB proteins and modulating E2F-driven transcriptional programs, p19 INK4D contributes to controlled proliferation, differentiation, and cellular senescence. CDKN2D is integrated into cell-cycle and stress-response networks, with functional relevance to oncogenic signaling contexts where CDK4/6–RB axis integrity influences growth control. Altered CDKN2D regulation has been explored in cancer biology and other hyperproliferative states to understand checkpoint bypass, lineage commitment, and genome stability.

p19 INK4D/CDKN2D CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CDKN2D expression without altering the underlying DNA sequence.

p19 INK4D/CDKN2D CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CDKN2D 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 CDKN2D transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous p19 INK4D/CDKN2D expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CDKN2D locus and enabling the study of p19 INK4D/CDKN2D-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of p19 INK4D/CDKN2D pathway restoration in tumor cells with silenced or reduced CDKN2D expression.

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