TGR5 CRISPR Activation Plasmid (h): sc-401875-ACT

Human GPBAR1 encodes TGR5, a bile acid–activated GPCR that couples primarily to Gαs to elevate intracellular cAMP and modulate downstream PKA/CREB signaling. TGR5 is expressed across metabolic and immune-relevant cell types and integrates signals from bile acids to regulate energy homeostasis, mitochondrial activity, and inflammatory tone. GPBAR1/TGR5 activity intersects with pathways controlling glucose and lipid metabolism, macrophage polarization, and barrier function in tissues exposed to bile acids. Dysregulated TGR5 signaling has been associated with metabolic dysfunction and chronic inflammatory processes, making it a useful target for mechanistic studies of GPCR signaling in hepatobiliary and gastrointestinal contexts.

TGR5 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous GPBAR1 expression without altering the underlying DNA sequence.

TGR5 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the GPBAR1 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 GPBAR1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous TGR5 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native GPBAR1 locus and enabling the study of TGR5-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of TGR5 pathway restoration in tumor cells with silenced or reduced GPBAR1 expression.

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