vinculin CRISPR Activation Plasmid (h): sc-400227-ACT

Human VCL encodes vinculin, a mechanosensitive actin-binding adaptor enriched at focal adhesions and adherens junctions where it couples integrin and cadherin complexes to the actin cytoskeleton. Vinculin regulates cell–ECM and cell–cell adhesion dynamics, force transmission, and cytoskeletal remodeling through pathways involving talin, paxillin, FAK/Src signaling, and Rho family GTPases. By tuning adhesion turnover and traction forces, VCL influences migration, polarity, and tissue morphogenesis, and its dysregulation is studied in contexts of altered invasiveness and metastatic potential. VCL-dependent mechanotransduction is also relevant to models of cardiomyocyte adhesion and contractile coupling, where changes in junctional integrity can impact stress responses.

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

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

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