The GOT2 Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal cell population derived from HEK293T, featuring targeted disruption of the GOT2 gene. This heterogeneous loss-of-function model enables pooled analysis of GOT2-dependent metabolic phenotypes without clonal selection bias, serving as a robust tool for studying mitochondrial aspartate aminotransferase function.
HEK293T cells, a derivative of HEK293, are human embryonic kidney cells expressing the SV40 large T antigen, widely used for recombinant protein expression, virus production, and gene editing. The SV40 T antigen enhances episomal plasmid replication, supporting high transfection efficiency and lentivirus generation, making this host suitable for generating polyclonal CRISPR knockout populations.
GOT2 encodes the mitochondrial isoform of aspartate aminotransferase, catalyzing the reversible transamination of oxaloacetate and glutamate to aspartate and ??-ketoglutarate. This reaction is central to the malate-aspartate shuttle, transferring reducing equivalents from cytosol to mitochondria and linking glycolysis to oxidative phosphorylation. GOT2 forms a homodimer and interacts with malate dehydrogenase MDH2, the oxoglutarate carrier SLC25A11, and the aspartate/glutamate carrier SLC25A12 to enable metabolite exchange. Its expression is regulated by upstream factors including PPARGC1A (PGC-1??), MYC, HIF1A, SIRT3, and mTORC1, integrating metabolic and growth signaling.
In HEK293T cells, which exhibit high glycolytic and glutaminolytic rates, GOT2 disruption offers a system to dissect the malate-aspartate shuttle??s role in redox balance and aspartate provision. Loss of GOT2 is expected to impair NAD+ regeneration and reduce aspartate availability, impacting proliferation and mitochondrial respiration. This polyclonal knockout model captures diverse editing outcomes, enabling the study of metabolic adaptations without clonal biases.
These cells support research in mitochondrial metabolism, cancer metabolic reprogramming, amino acid tracing, redox homeostasis, and neurodegeneration. Assays include Western blotting and RT-qPCR for knockout validation, enzymatic activity measurements, Seahorse respirometry, LC-MS-based metabolite profiling, and 13C-glutamine stable isotope tracing. Co-immunoprecipitation of GOT2 and proliferation assays under glutamine limitation are also applicable. For further information, contact Ascent Research.
