The KEAP1 Knockout DLD-1 Polyclonal Cells constitute a CRISPR/Cas9-edited polyclonal knockout cell population in which the KEAP1 gene has been disrupted to create a loss-of-function model of the human KEAP1 protein. This product provides a heterogeneous pool of DLD-1 cells carrying diverse KEAP1-targeting edits, enabling the study of KEAP1-dependent processes without the clonal artifacts inherent in single-cell-derived lines. The polyclonal format preserves the genetic diversity of the knockout population, offering a robust platform for bulk biochemical and functional assays in the context of colorectal adenocarcinoma.
The parental DLD-1 cell line, derived from a male patient with Duke’s type C colorectal adenocarcinoma, is a widely used model for human colorectal cancer. These epithelial cells harbor well-characterized driver mutations in APC, KRAS, TP53, and PIK3CA, and display high-level microsatellite instability (MSI-H). DLD-1 cells exhibit adherent epithelial morphology and retain key features of the original tumor, making them suitable for investigating signaling networks that intersect with oncogenic pathways. The genetic background of DLD-1 provides a relevant context for evaluating the functional impact of KEAP1 disruption on colorectal cancer biology.
KEAP1 functions as a substrate adaptor for the CUL3-RBX1 E3 ubiquitin ligase complex, which under basal conditions promotes the ubiquitination and proteasomal degradation of the transcription factor NRF2 (NFE2L2). In response to oxidative or electrophilic stress, critical cysteine residues in KEAP1 are modified by reactive oxygen species or electrophiles, leading to a conformational change that impairs NRF2 ubiquitination. This allows NRF2 to accumulate, translocate to the nucleus, and activate transcription of cytoprotective genes containing antioxidant response elements (AREs), such as HMOX1, NQO1, GCLC, GCLM, TXN, and PRDX1. KEAP1 activity is modulated by upstream regulators including PKC, p62/SQSTM1, and DPP3, and it interacts with proteins like PGAM5, IKK??, and PALB2. The KEAP1-NRF2 axis is a central node in the oxidative stress response and intersects with the ubiquitin-proteasome system, autophagy-lysosome pathway, and pentose phosphate pathway.
In DLD-1 colorectal adenocarcinoma cells, KEAP1 loss of function leads to constitutive NRF2 stabilization and upregulation of antioxidant and metabolic gene programs, which can influence tumor cell survival, proliferation, and drug resistance. The DLD-1 background, with its mutated APC, KRAS, TP53, and PIK3CA alleles, offers a unique setting to explore how KEAP1 deficiency modulates oncogenic signaling and stress adaptation. This knockout model is particularly valuable for dissecting the role of NRF2-mediated cytoprotection in colorectal cancer, as NRF2 hyperactivation has been implicated in chemoresistance and metabolic reprogramming. By providing a polyclonal KEAP1 knockout pool, researchers can investigate pathway dynamics in a population that more closely mimics the heterogeneous responses seen in tumors.
The KEAP1 Knockout DLD-1 Polyclonal Cells are suitable for a broad range of experimental applications, including investigating NRF2-mediated drug resistance mechanisms, characterizing the dynamics of KEAP1-NRF2 signaling under oxidative stress, and evaluating synthetic lethality interactions with KEAP1 loss. Representative assays that can be performed with this model include western blotting and RT-qPCR to assess NRF2 target gene expression, NRF2 luciferase reporter assays, flow cytometry for reactive oxygen species (ROS) levels, colony formation assays, drug sensitivity assays (e.g., cisplatin, 5-fluorouracil), co-immunoprecipitation to probe KEAP1 interaction partners, and glutathione level measurements. Additional applications encompass screening of NRF2 pathway inhibitors and metabolic profiling. For further details or technical support, please contact Ascent Research.
