GPT2 CRISPR Activation Plasmid (h): sc-403739-ACT

Human GPT2 (glutamic pyruvate transaminase 2; mitochondrial alanine aminotransferase) catalyzes reversible transamination between alanine and α-ketoglutarate to generate pyruvate and glutamate, linking amino acid metabolism to mitochondrial carbon flux. By supporting anaplerosis and glutamate handling, GPT2 contributes to nitrogen balance and energy homeostasis, influencing pathways connected to the TCA cycle and redox state. Altered GPT2 activity has been investigated in contexts of metabolic rewiring and mitochondrial dysfunction, where shifts in alanine–pyruvate interconversion can affect cellular proliferation and stress responses. As a mitochondrial enzyme, GPT2 is also relevant to studies of tissue-specific metabolism and nutrient utilization programs.

GPT2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous GPT2 expression without altering the underlying DNA sequence.

GPT2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the GPT2 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 GPT2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous GPT2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native GPT2 locus and enabling the study of GPT2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of GPT2 pathway restoration in tumor cells with silenced or reduced GPT2 expression.

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