Smad3 CRISPR Activation Plasmid (h): sc-400069-ACT

SMAD3 encodes Smad3, a receptor-regulated SMAD that transduces canonical TGF-β superfamily signals from activated type I receptors to the nucleus. Following phosphorylation, Smad3 forms complexes with SMAD4 and cooperates with lineage-specific transcription factors to control genes involved in extracellular matrix remodeling, epithelial–mesenchymal transition, immune regulation, and cell-cycle control. Through this transcriptional program, Smad3 influences processes such as fibrosis, inflammation, and differentiation across multiple tissue contexts. Dysregulated SMAD3 signaling has been linked to heritable connective tissue and vascular phenotypes, as well as aberrant TGF-β–driven programs in cancer and chronic inflammatory disease models.

Smad3 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SMAD3 expression without altering the underlying DNA sequence.

Smad3 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SMAD3 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 SMAD3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Smad3 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SMAD3 locus and enabling the study of Smad3-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Smad3 pathway restoration in tumor cells with silenced or reduced SMAD3 expression.

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