The ADI1 Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal population derived from HEK293T, with disruption of the ADI1 gene. This heterogeneous pool enables loss-of-function studies without clonal selection, supporting investigation of ADI1??s dual roles in methionine salvage and p53 regulation. The CRISPR-mediated gene disruption provides a flexible model for analyzing ADI1-dependent signaling in a high-transfection-efficiency background.
HEK293T, a derivative of HEK293 human embryonic kidney cells, stably expresses the SV40 large T antigen, promoting episomal plasmid replication and enabling high-level protein expression and viral production. Its epithelial origin and adenovirus transformation facilitate studies of cell cycle, apoptosis, and signal transduction, particularly p53 pathways, noting that SV40 large T antigen functionally inactivates endogenous p53, reducing background activity in downstream assays.
The ADI1 enzyme functions in the methionine salvage pathway by converting acireductone to KMTB, a methionine precursor. Additionally, ADI1 binds MDM2, inhibiting its ubiquitin ligase activity toward p53, resulting in p53 stabilization and activation of pro-apoptotic target genes such as BAX and PUMA. p53 transcriptionally upregulates ADI1, forming a feedback loop. ADI1 interacts with MTAP and integrates signals from methionine deprivation and polyamine metabolism, linking nutrient status to apoptosis.
In HEK293T, where p53 is inactivated, ADI1 knockout disrupts a mechanism that could counter SV40 large T antigen-mediated p53 suppression, allowing dissection of MDM2-p53 interactions and apoptosis. This model is relevant for cancers with methionine dependency, such as prostate and hepatocellular carcinomas. The polyclonal nature preserves heterogeneity, enabling studies of varied responses to methionine deprivation or MDM2 inhibitors and assessment of p53 target gene expression and caspase activation.
Applications include western blotting for p53, MDM2, and cleaved caspase-3; RT-qPCR for ADI1 and p53 targets; co-immunoprecipitation of ADI1-MDM2; cell viability and Annexin V apoptosis assays under methionine deprivation or stress; and p53 luciferase reporter assays. These cells facilitate cancer biology, tumor suppressor, and apoptosis research. The ADI1 Knockout HEK293T Polyclonal Cells are a valuable tool for probing ADI1??s metabolic and tumor-suppressive functions. For further information, contact Ascent Research.
