The ATG3 Knockout HEK293T Polyclonal Cells represent a CRISPR/Cas9-edited polyclonal knockout cell population engineered for the disruption of the autophagy-related gene ATG3 in the HEK293T host cell line. This pooled knockout model provides a powerful loss-of-function tool for dissecting the molecular mechanisms of autophagy without the constraints of monoclonal selection, enabling researchers to study gene function within a heterogeneous population that closely mimics natural genetic variation. The product is designed for advanced autophagy research and drug screening applications requiring robust and reproducible target gene disruption.
The HEK293T cell line, derived from human embryonic kidney cells, exhibits an adherent epithelial morphology and stably expresses the SV40 large T-antigen, which facilitates high transfection efficiency and robust protein expression. These features make HEK293T a favored host for viral vector production, transient protein overexpression, and lentiviral packaging. The integration of the large T-antigen also permits episomal replication of plasmids containing the SV40 origin, enhancing yield in various molecular biology applications.
ATG3 encodes an E2-like enzyme that plays an obligatory role in the ubiquitin-like conjugation pathway required for autophagosome biogenesis. It functions downstream of ATG7, which acts as an E1-activating enzyme, and cooperates with the ATG12-ATG5-ATG16L1 complex, which provides E3-like activity, to conjugate phosphatidylethanolamine (PE) to members of the ATG8 family, including MAP1LC3A, MAP1LC3B, GABARAP, and GABARAPL1. This lipidation step is essential for autophagosome membrane elongation and maturation. ATG3 activity is regulated by upstream nutrient-sensing kinases mTORC1 and AMPK, as well as by ULK1 and FOXO transcription factors, while ATG4B-mediated deconjugation and ATG7 interaction further modulate the cycle.
Disruption of ATG3 in HEK293T cells generates a model system in which autophagosome formation and mitochondrial homeostasis are compromised, making it highly suitable for investigating bulk and selective autophagy, including mitophagy and protein quality control. The high transfectability of HEK293T cells allows easy complementation with wild-type or mutant ATG3 constructs to validate phenotype?Cgenotype relationships, and the SV40 T-antigen supports high-level expression of introduced vectors, facilitating mechanistic studies.
This knockout product enables a wide array of assays, including monitoring autophagic flux via LC3-I/II conversion and p62 degradation by Western blotting, visualizing LC3 puncta formation by immunofluorescence, and assessing cell viability under nutrient deprivation or mitochondrial stress. It is particularly valuable for research areas such as neurodegeneration, oncology, infectious diseases, and host?Cpathogen interactions. Co-immunoprecipitation can assess ATG8 lipidation, providing a direct measure of ATG3 activity. For further information or technical support, please contact Ascent Research.
