PEPT1 CRISPR Activation Plasmid (h): sc-401259-ACT

SLC15A1 encodes the proton-coupled oligopeptide transporter PEPT1, a major apical membrane uptake system in intestinal epithelial cells that imports di- and tripeptides and numerous peptidomimetic substrates. PEPT1 couples substrate transport to the transmembrane H+ gradient, linking nutrient absorption to cellular pH homeostasis and metabolic integration of amino acid availability. Through its role in epithelial barrier physiology and nutrient-sensing processes, altered PEPT1 expression has been studied in gastrointestinal inflammation and dysregulated intestinal transport phenotypes. In vitro, PEPT1 is frequently used to model epithelial uptake mechanisms, transporter regulation by inflammatory cues, and substrate competition in gut-relevant systems.

PEPT1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SLC15A1 expression without altering the underlying DNA sequence.

PEPT1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SLC15A1 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 SLC15A1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous PEPT1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SLC15A1 locus and enabling the study of PEPT1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of PEPT1 pathway restoration in tumor cells with silenced or reduced SLC15A1 expression.

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