GPT CRISPR Activation Plasmid (h): sc-402909-ACT

Human GPT encodes alanine aminotransferase 2, a mitochondrial enzyme that catalyzes reversible transamination between alanine and α-ketoglutarate to generate pyruvate and glutamate. Through this reaction, GPT helps couple amino acid utilization to the TCA cycle, anaplerosis, and nitrogen handling, supporting mitochondrial bioenergetics and redox balance. GPT activity is linked to broader metabolic programs that influence gluconeogenic flux, lipid handling, and cellular responses to nutrient availability. Dysregulated transaminase-associated metabolism is frequently observed in metabolic stress states and cancer-associated rewiring of mitochondrial carbon and nitrogen flow, making GPT a useful node for studying context-dependent metabolic vulnerabilities.

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

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

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