AATM CRISPR Activation Plasmid (h): sc-403969-ACT

Human GOT2 encodes mitochondrial aspartate aminotransferase (AATM), a pyridoxal phosphate–dependent enzyme that catalyzes reversible transamination between glutamate/oxaloacetate and aspartate/α-ketoglutarate. AATM is a core component of the malate–aspartate shuttle, coupling cytosolic and mitochondrial redox balance by transferring reducing equivalents into mitochondria and supporting NADH/NAD⁺ homeostasis. Through its roles in amino acid metabolism, TCA anaplerosis, and nitrogen handling, GOT2 influences mitochondrial bioenergetics and aspartate availability required for nucleotide synthesis. Dysregulation of GOT2-linked pathways has been associated with altered metabolic states observed in proliferative and stress-adapted cells, making it relevant to studies of metabolic rewiring and mitochondrial function.

AATM CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous GOT2 expression without altering the underlying DNA sequence.

AATM CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the GOT2 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 GOT2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous AATM expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native GOT2 locus and enabling the study of AATM-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of AATM pathway restoration in tumor cells with silenced or reduced GOT2 expression.

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