Nodal CRISPR Activation Plasmid (h): sc-401439-ACT

Human NODAL encodes the secreted morphogen Nodal, a TGF-β superfamily ligand that signals through activin receptors and SMAD2/3 to regulate germ layer specification, axis formation, and maintenance of developmental transcriptional programs. Nodal activity is tightly controlled by feedback with antagonists such as LEFTY proteins and by cross-talk with WNT and FGF pathways, shaping cell fate decisions and epithelial–mesenchymal transitions. Aberrant NODAL pathway reactivation has been associated with altered differentiation states and invasive phenotypes in multiple tumor contexts, supporting its use as a mechanistic node in developmental and disease models. As a pathway ligand, Nodal provides a tractable readout for studying paracrine signaling, SMAD-dependent transcription, and lineage plasticity in human cell systems.

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

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

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