This product consists of CRISPR/Cas9-edited polyclonal ANKRD16 knockout HT29 cells, providing a genetically heterogeneous population with targeted disruption of the ANKRD16 locus. The polyclonal format avoids clonal selection artifacts and enables study of gene function across a diverse cellular background derived from the HT29 colorectal adenocarcinoma line. Loss of ANKRD16 protein expression in these cells is generated using CRISPR/Cas9-mediated gene disruption, resulting in a loss-of-function model suitable for investigating the regulatory roles of this chaperone-like factor in stress signaling.
The host cell line HT29 is a widely used human colorectal adenocarcinoma epithelial model, known for its adherent growth properties and well-characterized tumorigenic phenotype. These cells harbor mutations in the tumor suppressors TP53 (p53) and APC, which are commonly altered in colorectal cancers. The genetic background of HT29 cells provides a relevant context for studying oncogenic signaling, epithelial biology, and therapeutic responses, particularly in pathways that intersect with cellular stress and survival mechanisms.
ANKRD16 functions as a chaperone-like regulator of the integrated stress response (ISR), primarily through its interaction with alanyl-tRNA synthetase (AARS). Under endoplasmic reticulum (ER) stress or other insults, ANKRD16 binds and stabilizes AARS, preventing its misfolding and aggregation. This activity suppresses the PERK-dependent phosphorylation of eIF2??, thereby attenuating the induction of the transcription factor ATF4 and downstream targets such as CHOP. Consequently, ANKRD16 dampens the ISR and the unfolded protein response, promoting cell survival. Its function is integrated within a signaling network involving upstream ER stress sensors, eIF2?? kinases, and downstream apoptotic regulators, highlighting its role as a molecular brake on stress-induced death pathways.
In the HT29 colorectal cancer context, ANKRD16 knockout provides a powerful tool to dissect how tumor cells manage proteotoxic stress. Given the background of p53 and APC mutations, loss of ANKRD16 may exacerbate sensitivity to pharmacological ER stressors such as tunicamycin, revealing dependencies on the ISR for viability. This model allows researchers to study the interplay between oncogenic signaling, the ISR, and apoptosis regulation, potentially uncovering vulnerabilities that can be exploited therapeutically in colorectal cancers and other malignancies.
Typical applications include detailed investigation of the integrated stress response, tRNA synthetase biology, and neurogenerative disease modeling. Researchers can employ western blotting to assess ANKRD16, phospho-eIF2??, and ATF4 levels, RT-qPCR analysis of ATF4 target genes, and stress challenge assays with tunicamycin. Cell viability can be monitored via MTT assays, and protein interactions examined through co-immunoprecipitation of AARS. Flow cytometry-based Annexin V apoptosis assays enable quantification of cell death pathways. For further technical support or customization, please contact Ascent Research.
