The ATG10 Knockout HEK293T Polyclonal Cells constitute a CRISPR/Cas9-edited polyclonal knockout cell population in which the autophagy-related gene ATG10 has been genetically disrupted. This polyclonal population, derived from HEK293T human embryonic kidney cells, offers a heterogeneous loss-of-function model for investigating the molecular requirements of the autophagy conjugation cascade. Due to the non-clonal nature of the knockout pool, it preserves the inherent cellular variability of a mass-edited population, making it particularly suitable for experiments that require representative polygenic effects rather than single-clone idiosyncrasies.
The parental HEK293T cell line is a widely utilized host for genetic perturbation studies, originally generated by adenovirus 5 transformation of human embryonic kidney epithelial cells and further modified to stably express the SV40 large T antigen. This immortalized background confers high transfection efficiency, robust exogenous protein expression, and rapid proliferation, rendering it an ideal platform for generating and expanding knockout populations. The epithelial origin of HEK293T cells also provides a relevant context for studying autophagy in epithelial biology, stress responses, and metabolic regulation.
ATG10 functions as an E2-like enzyme essential for the covalent conjugation of the ubiquitin-like protein ATG12 to ATG5, a core event in the elongation phase of autophagosome formation. This reaction is orchestrated by the E1-like enzyme ATG7 and results in the irreversible formation of the ATG12?CATG5 conjugate, which subsequently assembles with ATG16L1 to form a large multimeric complex. The ATG12?CATG5?CATG16L1 complex localizes to the isolation membrane and directs the lipidation and membrane association of LC3/GABARAP proteins, a step crucial for autophagosome expansion and cargo sequestration. Upstream, ATG10-mediated conjugation is regulated by nutrient-sensing kinases such as AMPK and mTORC1, which respectively activate or inhibit the ULK1 complex and downstream autophagy initiation factors including TFEB. In this knockout model, ATG10 disruption abolishes ATG12?CATG5 formation, thereby preventing assembly of the ATG12?CATG5?CATG16L1 complex and blocking LC3 lipidation, ultimately impairing autophagosome biogenesis.
In the HEK293T context, loss of ATG10 provides a genetically defined system to dissect autophagy-dependent processes without confounding off-target effects often associated with pharmacological inhibitors. Because autophagy is a key cellular response to starvation, proteotoxic stress, and pathogen invasion, this knockout model is particularly valuable for studying mechanisms where autophagy intersects with cancer cell survival, neurodegenerative protein aggregation, or host?Cpathogen interactions. The rapid growth and ease of manipulation of HEK293T cells facilitate high-throughput screens and transient rescue experiments, allowing researchers to link ATG10 function directly to autophagic flux and downstream cellular phenotypes.
Typical applications include monitoring autophagy flux by western blotting for LC3-II and p62/SQSTM1 levels following lysosomal inhibitor treatment, visualizing autophagosome puncta via immunofluorescence, and assessing cell viability under nutrient starvation. This polyclonal knockout pool also enables co-immunoprecipitation studies to examine the integrity of the ATG12?CATG5 interaction and RT-qPCR analysis of autophagy gene expression. Additionally, the cells can be exploited in drug sensitivity screens to identify autophagy modulators relevant to cancer, neurodegeneration, or infectious disease. For further inquiries regarding this product, please contact Ascent Research.
