GPAM CRISPR Activation Plasmid (h): sc-403961-ACT

Human GPAM (glycerol-3-phosphate acyltransferase, mitochondrial) catalyzes the initial and rate-limiting step of de novo glycerolipid synthesis by converting glycerol-3-phosphate and long-chain acyl-CoA into lysophosphatidic acid. This enzymatic activity links mitochondrial lipid handling to phosphatidic acid production, triacylglycerol assembly, and broader phospholipid remodeling, thereby influencing membrane biogenesis and lipid droplet formation. GPAM functions at the intersection of fatty acid utilization and energy homeostasis, with downstream impacts on signaling lipids and metabolic network balance. Altered GPAM expression or activity has been associated with dysregulated lipid metabolism phenotypes relevant to hepatic steatosis, insulin resistance, and cardiometabolic risk pathways, making it a useful node for mechanistic studies in metabolic biology.

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

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

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