The KYNU Knockout HT29 Polyclonal Cells comprise a CRISPR/Cas9-edited polyclonal knockout cell population generated from the HT29 human colorectal adenocarcinoma cell line, featuring disruption of the KYNU gene. This pool provides a genetically heterogeneous model of KYNU loss-of-function, enabling robust functional studies without the constraints of clonal selection.
HT29 cells are epithelial in origin, isolated from a primary colorectal adenocarcinoma in a 44-year-old female patient. These cells are widely employed as a model of intestinal epithelial cells, retaining the capacity to differentiate under appropriate culture conditions, and serve as a relevant system for investigating colorectal cancer biology, barrier function, and metabolic regulation.
KYNU encodes kynureninase, a pyridoxal phosphate-dependent hydrolase that cleaves kynurenine and 3-hydroxykynurenine into anthranilic acid and 3-hydroxyanthranilic acid, respectively, within the kynurenine pathway. This pathway is initiated by IDO1 and TDO2, upregulated by IFN-?? and TNF-?? via STAT1 and NF-??B. KYNU functions downstream of KMO and upstream of HAAO, generating quinolinic acid, picolinic acid, and NAD+. Metabolites like kynurenine and 3-hydroxykynurenine promote T-cell suppression, linking tryptophan catabolism to immune regulation.
Disruption of KYNU in HT29 cells ablates kynureninase activity, causing accumulation of kynurenine and 3-hydroxykynurenine while depleting 3-hydroxyanthranilic acid and its downstream metabolites. This metabolic blockade impairs NAD+ biosynthesis and alters the profile of immunomodulatory molecules within the tumor microenvironment. Given the responsiveness of HT29 cells to inflammatory cytokines and their capacity for differentiation, this model offers a physiologically pertinent context to explore how KYNU-mediated tryptophan metabolism influences colorectal cancer cell fitness, immune evasion, and cellular energetics.
These polyclonal knockout cells are ideally suited for a variety of research applications, including dissection of the kynurenine pathway, cancer immunometabolism, and NAD+ homeostasis. Applicants may employ LC-MS metabolomics to quantify metabolic intermediates, RT-qPCR or western blotting to confirm gene disruption, and functional assays such as NAD+/NADH ratio measurement, ELISA for kynurenine, immunofluorescence, and cell viability or migration assays to evaluate phenotypic outcomes. This model is especially useful for studies examining the intersection of metabolic enzymes and immune regulation in the colorectal cancer context. For additional information or technical support, please contact Ascent Research.
