The ATG2B Knockout HAP1 Polyclonal Cells provide a CRISPR/Cas9-edited polyclonal knockout cell population in which the autophagy-related gene ATG2B has been disrupted. This polyclonal knockout model serves as a robust loss-of-function tool for investigating ATG2B-dependent mechanisms in autophagy and lipid transfer, without the need for single-cell clone isolation. The knockout cell population is derived from the HAP1 cell line and is suitable for a wide range of functional and phenotypic assays.
HAP1 is a near-haploid human fibroblast-like cell line originally derived from the KBM-7 chronic myeloid leukemia (CML) cell line. Its haploid karyotype eliminates the complexity of heterozygous alleles, enabling straightforward genotype-to-phenotype correlation and making it an ideal host for genetic perturbation studies. Widely used in haploid genetic screens, HAP1 cells have been instrumental in identifying genes essential for autophagy and other pathways, providing a clean genetic background for knockout studies.
ATG2B is a critical autophagy protein that mediates the transfer of lipids from the endoplasmic reticulum to the expanding phagophore, driving autophagosome biogenesis. This process is tightly regulated by upstream nutrient-sensing pathways: under starvation, mTORC1 is inhibited, allowing AMPK and the ULK1 complex (ULK1, ATG13, FIP200, ATG101) to activate the autophagy machinery. ATG2B functions in a complex with WIPI1 and WIPI2, and its lipid-transfer activity is coordinated with the ATG9A scramblase. Downstream, ATG2B promotes the lipidation of LC3 and the formation of autophagosomes, leading to the engulfment and degradation of cargo receptors such as p62/SQSTM1. Consequently, ATG2B disruption impairs autophagic flux and compromises cellular homeostasis.
In the HAP1 background, ATG2B knockout provides a unique platform to dissect autophagy regulation within a CML-derived cellular environment. The haploid nature simplifies genetic complementation experiments and enhances the precision of functional studies, while the leukemic origin allows exploration of autophagy??s role in cancer cell survival, proliferation, and drug resistance. This model is particularly valuable for conducting synthetic lethality screens and for interrogating genetic interactions that may uncover therapeutic vulnerabilities in autophagy-dependent cancers.
These polyclonal knockout cells are widely applicable in autophagy research. Key assay endpoints include Western blotting for LC3-II and p62 to assess autophagic flux, immunofluorescence imaging of WIPI2 and LC3 puncta to monitor autophagosome formation, and autophagy flux assays using Bafilomycin A1 to block lysosomal degradation. Co-immunoprecipitation studies can be performed to examine ATG2B interactions with WIPI isoforms and ATG9A, while lipidomics analysis enables profiling of lipid transfer dynamics. Additionally, the cells are suitable for drug screening of autophagy modulators and for genetic interaction studies. For additional information or technical support, please contact Ascent Research.
