Villin CRISPR Activation Plasmid (h): sc-400587-ACT

VIL1 encodes villin, an actin-binding and actin-severing protein enriched in intestinal and renal epithelial microvilli, where it helps establish and remodel the apical brush border. Villin regulates cytoskeletal organization, epithelial polarity, and cell motility through Ca²⁺-dependent control of F-actin bundling and severing, linking membrane dynamics to differentiation programs in absorptive epithelia. As a canonical marker of enterocyte lineage, altered VIL1 expression is frequently used to track epithelial state changes during inflammation, injury responses, and oncogenic transformation. These properties make villin a useful entry point for studying microvillar architecture, barrier biology, and cytoskeleton-dependent signaling in human epithelial models.

Villin CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous VIL1 expression without altering the underlying DNA sequence.

Villin CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the VIL1 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 VIL1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Villin expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native VIL1 locus and enabling the study of Villin-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Villin pathway restoration in tumor cells with silenced or reduced VIL1 expression.

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