This product consists of a CRISPR/Cas9-edited polyclonal knockout cell population in which the human ATG3 gene has been disrupted in the HAP1 cell background. It provides a versatile loss-of-function model to investigate autophagy, a catabolic pathway relevant to neurodegeneration, cancer, and infection. The polyclonal format ensures a heterogeneous knockout pool, minimizing clonal selection biases and enabling robust population-based functional studies.
The HAP1 cell line is a near-haploid human line derived from the KBM-7 chronic myeloid leukemia patient, male. Its haploid genome greatly facilitates CRISPR-mediated gene targeting and phenotypic analysis, making it an exemplary host for haploid genetic screens and leukemia-specific pathway interrogation. The cell line retains characteristics of its leukemic origin, offering context for dissecting cancer-relevant autophagy functions.
ATG3 acts as an E2-like enzyme central to the ATG8 lipidation cascade, an ubiquitylation-like reaction that conjugates LC3 and GABARAP family proteins to phosphatidylethanolamine on the autophagosomal membrane. This process is tightly regulated by upstream nutrient-sensing kinases: AMPK and mTORC1 via the ULK1 complex, and is induced by starvation or rapamycin. The ATG3 enzyme functions in concert with the E1-like ATG7 and the E3-like ATG12?CATG5?CATG16L1 complex, directly interacting with ATG7 and the ATG12?CATG5 conjugate to transfer LC3. ATG3-mediated lipidation is indispensable for phagophore elongation and autophagosome closure. Consequently, knockout of ATG3 ablates LC3-II formation and blocks autophagy.
In the HAP1 context, ATG3 knockout yields a complete autophagy disruption that synergizes with the haploid genetic background to unmask recessive phenotypes. This model enables precise dissection of autophagy??s roles in leukemia cell survival, metabolic stress responses, and drug resistance, while minimizing confounding effects from diploid gene redundancy. The near-haploid state also facilitates arrayed and pooled functional genomics screens aimed at identifying genetic or chemical modifiers.
These polyclonal cells are well-suited for standard autophagy assays, including western blotting to confirm loss of LC3-II, immunofluorescence to visualize LC3 puncta, and flux assessments using bafilomycin A1. Researchers can further explore ATG3-dependent effects on cell viability under nutrient deprivation, perform transcriptomic profiling by RNA-seq, and conduct combinatorial treatments with autophagy modulators such as rapamycin. The heterogeneous knockout population is particularly valuable for high-throughput screening campaigns to uncover novel autophagy regulators or synthetic lethal partners. For technical inquiries and experimental support, contact Ascent Research.
