The GOT1 knockout HT29 polyclonal cell product is a heterogeneous population of HT29 human colorectal adenocarcinoma cells harboring CRISPR/Cas9-mediated disruption of the GOT1 gene. Generated by introducing Cas9 nuclease and guide RNAs targeting GOT1, this polyclonal pool is enriched for edited cells while maintaining the genetic diversity of the targeted population. It serves as a robust loss-of-function model for studying GOT1-dependent metabolic and signaling processes without the biases of clonal selection.
HT29 cells, derived from a 44-year-old female patient??s primary colorectal adenocarcinoma, exhibit adherent epithelial morphology and a mutant p53 background. This well-characterized line is widely used as a colon cancer model to study epithelial barrier function, mucin production, and drug transport, offering a physiologically relevant context for examining the metabolic roles of GOT1 in colorectal tumorigenesis.
GOT1 encodes cytosolic aspartate aminotransferase, catalyzing the reversible transamination of aspartate and ??-ketoglutarate to oxaloacetate and glutamate. A core enzyme of the malate-aspartate shuttle, it cooperates with mitochondrial GOT2, malate dehydrogenase 1 (MDH1), and citrate synthase, and its activity is facilitated by the mitochondrial carriers SLC25A11 and SLC25A12. Transcription of GOT1 is regulated by c-Myc, HIF-1??, ATF4, NRF2, and p53 in a context-dependent manner. Downstream, GOT1 modulates the levels of aspartate, oxaloacetate, and glutamate, thereby influencing nucleotide biosynthesis, TCA cycle intermediates, and glutathione synthesis for redox balance.
Knocking out GOT1 in HT29 cells impairs the malate-aspartate shuttle, reducing mitochondrial NADH transfer and aspartate-driven nucleotide biosynthesis. This disruption fosters redox stress and attenuates proliferation under nutrient-limited conditions, mimicking the tumor microenvironment. The HT29 cell??s glutamine dependency and p53 mutation may accentuate sensitivity to GOT1 loss, offering a platform to explore metabolic vulnerabilities and adaptive mechanisms in colorectal cancer.
This polyclonal knockout model supports diverse assays: LC-MS metabolomics for aspartate, TCA intermediates, and glutathione; 13C-glutamine tracing for flux analysis; and immunoblotting or RT-qPCR for GOT1 knockdown confirmation. Functional readouts include proliferation (MTS/MTT), apoptosis (Annexin V/PI), ROS detection (DCFDA), colony formation, and drug sensitivity tests with glutaminase inhibitors like CB-839. It is instrumental for validating GOT1 as a metabolic target in colorectal cancer. For further details, contact Ascent Research.
