Smad4 CRISPR Activation Plasmid (h): sc-400110-ACT

SMAD4 encodes Smad4, a central transcriptional co-mediator of the TGF-β/BMP signaling network that forms complexes with receptor-regulated SMADs to regulate context-dependent gene expression programs. Through these complexes, Smad4 controls processes including cell cycle regulation, differentiation, epithelial–mesenchymal dynamics, and extracellular matrix remodeling. Perturbation of SMAD4-dependent transcription is linked to dysregulated growth control and altered tissue homeostasis, with frequent genetic or functional inactivation reported across multiple tumor types. SMAD4 status is also used to study pathway crosstalk between TGF-β signaling and MAPK, PI3K–AKT, and Wnt programs that collectively shape transcriptional outputs.

Smad4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SMAD4 expression without altering the underlying DNA sequence.

Smad4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SMAD4 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 SMAD4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Smad4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SMAD4 locus and enabling the study of Smad4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Smad4 pathway restoration in tumor cells with silenced or reduced SMAD4 expression.

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