Cdk4 CRISPR Activation Plasmid (h): sc-400148-ACT

Human CDK4 encodes cyclin-dependent kinase 4 (Cdk4), a core regulator of G1 phase progression that partners with D-type cyclins to phosphorylate RB family proteins and promote E2F-dependent transcription required for S-phase entry. CDK4 activity is integrated with mitogenic signaling pathways, including receptor tyrosine kinase–RAS–MAPK and PI3K–AKT axes, and is modulated by CDK inhibitors such as CDKN2A (p16INK4A). Dysregulated CDK4 signaling perturbs cell-cycle checkpoint control and contributes to aberrant proliferation programs relevant to cancer biology, while also influencing lineage decisions and metabolic remodeling in proliferating cells. As a nodal kinase within the cyclin D–CDK4/6–RB pathway, CDK4 serves as a widely used molecular handle for interrogating growth control and cell-cycle coupling to transcription.

Cdk4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CDK4 expression without altering the underlying DNA sequence.

Cdk4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CDK4 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 CDK4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Cdk4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CDK4 locus and enabling the study of Cdk4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Cdk4 pathway restoration in tumor cells with silenced or reduced CDK4 expression.

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