The ATG16L1 Knockout HT29 Polyclonal Cells product comprises a CRISPR/Cas9-edited polyclonal knockout cell population derived from the human HT29 colorectal adenocarcinoma cell line, engineered to disrupt the autophagy-related gene ATG16L1. This loss-of-function model is generated through CRISPR/Cas9-mediated gene disruption, yielding a heterogeneous pool of cells carrying targeted mutations in the ATG16L1 locus, facilitating robust analysis of autophagy pathway dependencies and inflammatory signaling in a physiologically relevant epithelial context.
HT29 cells are a widely characterized human female colorectal adenocarcinoma epithelial line established from a primary tumor. These mucin-producing cells serve as a well-validated intestinal epithelial barrier model, exhibiting features of enterocyte differentiation. Their adenocarcinoma origin and epithelial morphology make them particularly suitable for dissecting mechanisms of intestinal cell biology, host-microbe interactions, and the molecular pathology of inflammatory bowel disease and colorectal cancer.
ATG16L1 encodes a core component of the ATG12-ATG5-ATG16L1 complex, which functions as an E3-like enzyme during autophagosome formation by conjugating LC3 (MAP1LC3B) to phosphatidylethanolamine on nascent phagophores. This activity is essential for autophagy flux, xenophagy, and LC3-associated phagocytosis. ATG16L1 is regulated by mTORC1, AMPK, and ULK1, and acts upstream of LC3 lipidation and p62/SQSTM1 degradation. It also interacts with ATG5, ATG12, WIPI2, NOD2, and RAB33B, and influences IL-1?? secretion and Paneth cell function. Transcription of ATG16L1 is modulated by TFEB, FOXO3, and inflammatory signals such as TNF-?? and IFN-??.
In the HT29 intestinal epithelial background, ATG16L1 disruption specifically impairs autophagy and xenophagy, mirroring functional defects observed with ATG16L1 variants linked to Crohn’s disease. Knockout cells exhibit accumulation of autophagy substrates like p62 and a block in LC3 lipidation, compromising intracellular bacterial clearance and altering pro-inflammatory cytokine secretion, particularly IL-1??. This model thus recapitulates key cellular phenotypes associated with inflammatory bowel disease susceptibility, enabling mechanistic studies of how autophagy defects contribute to epithelial barrier dysfunction, aberrant immune responses, and pathogenesis of colitis-associated carcinoma.
Researchers can employ this polyclonal knockout population in diverse applications, including Western blotting for LC3-I/II and p62, autophagy flux assays with chloroquine, immunofluorescence for LC3 puncta, co-immunoprecipitation of autophagy complexes, and bacterial invasion/clearance assays. The model is also suitable for IL-1?? ELISA, RT-qPCR profiling of autophagy-related genes, and viability assays under nutrient stress. These readouts support investigation of autophagy defects in inflammatory bowel disease, screening of autophagy modulators, and studies of host-pathogen interactions, particularly with Salmonella. For additional product information and technical support, please contact Ascent Research.
