The ABCG2 Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population in which the ABCG2 gene has been disrupted to generate a heterogeneous loss-of-function model. This polyclonal pool contains a diverse array of cells harboring various CRISPR-induced alleles, offering a robust system for studying ABCG2-dependent processes without the biases associated with single-cell clones. The knockout abolishes the expression of the ABCG2 efflux transporter, enabling researchers to dissect its contributions to drug resistance, metabolite transport, and cellular detoxification pathways.
HAP1 is a near-haploid human CML cell line derived from KBM-7, featuring the BCR-ABL1 fusion and a male genetic background. Growing in suspension, these cells are a mainstay of haploid genetic screens due to their single-copy genome, which accentuates knockout phenotypes. The constitutive BCR-ABL kinase activity makes HAP1 a clinically relevant model for studying tyrosine kinase inhibitor sensitivity and resistance.
ABCG2 is a homodimeric ABC transporter that extrudes numerous xenobiotics and endogenous metabolites. Its expression is controlled by nuclear receptors (PXR, CAR, AhR), the NRF2/ARE oxidative stress pathway, and microRNAs (miR-328, miR-519c). The transporter localizes to the apical membrane via PDZK1, is phosphorylated by PIM1, and undergoes caveolin-1-dependent endocytosis. Key substrates include chemotherapeutics (mitoxantrone, topotecan, imatinib), the dye Hoechst 33342, and metabolites such as urate and porphyrins, linking it to multidrug resistance and the stem cell side population.
In HAP1 cells, ABCG2 knockout removes a critical factor in drug resistance, especially to tyrosine kinase inhibitors like imatinib. The polyclonal nature avoids clonal artifacts and provides a representative loss-of-function landscape. Combined with the near-haploid background, this model simplifies dissecting ABCG2 contributions to BCR-ABL signaling, NRF2-mediated stress responses, and nuclear receptor-driven metabolism. Researchers can examine how ABCG2 polymorphisms influence substrate specificity, yielding insights for pharmacogenetics, gout, and porphyria.
These knockout cells support efflux assays with mitoxantrone or Hoechst 33342 and flow cytometry to quantify ABCG2 activity and side populations. Drug sensitivity testing (imatinib, topotecan) elucidates resistance mechanisms, while western blotting and RT-qPCR verify ABCG2 loss. Co-immunoprecipitation can probe homodimerization or PDZK1 binding, and ATPase assays measure transport kinetics. Transwell models enable vectorial drug transport studies. Additionally, they facilitate pharmacogenetic profiling of ABCG2 variants and CRISPR-based modifier screens. For detailed protocols, contact Ascent Research.
