ITF CRISPR Activation Plasmid (h2): sc-416266-ACT-2

Human TFF3 (trefoil factor 3) encodes the secreted peptide ITF, a mucin-associated trefoil factor highly expressed in intestinal and other mucosal epithelia where it supports barrier integrity by promoting epithelial restitution, migration, and wound-adjacent repair responses. TFF3/ITF interacts with mucus components and modulates signaling linked to cytoskeletal remodeling and survival programs, including MAPK/ERK and PI3K/AKT-associated processes, thereby influencing cell–cell junctional maintenance and inflammatory stress adaptation. Dysregulated TFF3 expression has been reported in contexts of mucosal injury and chronic inflammation and is frequently studied in gastrointestinal disorders as well as epithelial cancers where it can correlate with altered differentiation, invasion, and metastasis-related phenotypes. Gene editing of TFF3 enables mechanistic dissection of mucosal repair pathways, epithelial–immune crosstalk, and secreted-factor contributions to organoid, barrier, and tumor microenvironment models.

ITF CRISPR Activation Plasmid (h2) provides a targeted, non-destructive approach to upregulating endogenous TFF3 expression without altering the underlying DNA sequence.

ITF CRISPR Activation Plasmid (h2) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the TFF3 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 TFF3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous ITF expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native TFF3 locus and enabling the study of ITF-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of ITF pathway restoration in tumor cells with silenced or reduced TFF3 expression.

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