nestin CRISPR Activation Plasmid (h): sc-400156-ACT

Human NES encodes nestin, a class VI intermediate filament protein enriched in neural stem/progenitor cells and other multipotent precursors, where it supports cytoskeletal organization during rapid proliferation, migration, and lineage commitment. Nestin expression is tightly linked to developmental programs and stress-responsive remodeling, intersecting with signaling networks such as Notch, Wnt/β-catenin, and growth factor pathways that regulate stemness and differentiation. In adult tissues, NES is often induced during injury and regeneration, reflecting roles in reactive glia, angiogenesis, and tissue remodeling. Dysregulated nestin expression is frequently used as a marker of poorly differentiated states and has been associated with tumor cell plasticity and invasiveness across multiple cancer contexts.

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

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

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