The GPX4 Knockout DLD-1 Polyclonal Cells consist of a CRISPR/Cas9-edited polyclonal population of DLD-1 human colorectal adenocarcinoma epithelial cells carrying disruption of the glutathione peroxidase 4 (GPX4) gene. This loss-of-function model is produced by electroporation of Cas9?Cguide RNA ribonucleoprotein complexes, yielding a heterogeneous pool of edited cells suitable for pooled functional studies without clonal selection.
DLD-1 is a colorectal adenocarcinoma cell line derived from a Duke’s type C patient, characterized by microsatellite instability (MSI-high), mutant APC, and a KRAS G13D activating mutation, alongside mismatch repair deficiency. These genetic attributes make it a relevant system for studying oncogenic signaling, DNA repair defects, and metabolic vulnerabilities in colorectal cancer.
GPX4 is a selenoenzyme that reduces phospholipid hydroperoxides to lipid alcohols using glutathione, thereby protecting membranes from oxidative damage and acting as a central negative regulator of ferroptosis. Its expression is controlled by upstream regulators including NFE2L2, selenium, ATF4, and TP53, and it functionally interacts with the selenocysteine incorporation machinery (SBP2, SECISBP2). In ferroptosis, GPX4 operates downstream of SLC7A11-mediated cystine import and glutathione synthesis, counteracting pro-ferroptotic factors such as ACSL4, LPCAT3, and ALOXs. Disruption of GPX4 leads to accumulation of lipid reactive oxygen species and ferroptotic cell death.
In the DLD-1 context, GPX4 knockout unveils ferroptosis susceptibility influenced by MSI-high status and mutant KRAS signaling. The model enables investigation of synthetic lethal interactions and the efficacy of ferroptosis inducers like RSL3 and erastin in a mismatch repair-deficient background, while also allowing dissection of NFE2L2-dependent antioxidant responses.
Applications include lipid peroxidation measurement via C11-BODIPY and MDA assays, western blotting, RT-qPCR, cell viability assays, and flow cytometry for lipid ROS. The cells support drug screening for ferroptosis modulators and mechanistic studies of selenoprotein biology. For more information, contact Ascent Research.
