The HPRT1 Knockout HAP1 Polyclonal Cells represent a CRISPR/Cas9-edited polyclonal knockout cell population generated by targeted disruption of the HPRT1 gene in the near-haploid HAP1 human cell line. This product provides a loss-of-function model for studying HPRT1-dependent purine salvage and nucleotide metabolism without requiring isolation of single-cell clones. The polyclonal format, produced by population-level editing with Cas9 and a gene-specific guide RNA, ensures broad representation of knockout alleles across a mixed cell pool, enabling robust functional genomics experiments. Researchers can immediately apply this model in assays such as 6-thioguanine resistance screening, where HPRT1-deficient cells survive cytotoxic selection, confirming effective gene disruption.
The HAP1 cell line is derived from the KBM-7 chronic myelogenous leukemia (CML) haploid cell line and retains a near-haploid karyotype in the majority of cells, making it uniquely suited for knockout studies. As a haploid model, HAP1 eliminates the confounding effects of a second allele, ensuring that a single CRISPR/Cas9-induced mutation can produce a complete loss of protein function across the population. Its CML origin also provides a cancer-relevant background for exploring how disruptions in purine metabolism influence tumor cell proliferation, drug sensitivity, and metabolic reprogramming. The cells grow in adherent culture and are compatible with standard cell biology techniques, including western blotting, immunocytochemistry, and high-throughput screening platforms.
HPRT1 encodes hypoxanthine-guanine phosphoribosyltransferase, a purine salvage enzyme that catalyzes the transfer of the phosphoribosyl moiety from PRPP to hypoxanthine and guanine, forming IMP and GMP, respectively. This housekeeping enzyme is transcriptionally regulated by the SP1 transcription factor and is feedback-modulated by intracellular purine nucleotide levels. HPRT1 forms a homotetramer and operates in concert with related purine metabolism components such as APRT, adenosine kinase, purine nucleoside phosphorylase, IMP dehydrogenase, and GMP synthase. Downstream, its activity directly sustains GTP and ATP pools essential for DNA and RNA synthesis. Genetic ablation of HPRT1 thus disrupts nucleotide homeostasis, leading to accumulation of phosphoribosylpyrophosphate (PRPP) and increased de novo purine synthesis, a phenotype observed in Lesch-Nyhan syndrome patients.
In the HAP1 context, HPRT1 knockout recapitulates the biochemical defects of Lesch-Nyhan syndrome, including complete loss of HPRT enzyme activity and hypersensitivity to purine analogs such as 6-thioguanine and 6-mercaptopurine. The haploid nature of HAP1 cells amplifies the penetrance of the knockout phenotype, making this model particularly sensitive for structure?Cfunction analyses and drug screening campaigns targeting purine pathways. Furthermore, the absence of a wild-type allele facilitates straightforward interpretation of metabolic flux studies using stable isotope tracing and HPLC-based metabolite profiling. This polyclonal knockout population is ideal for investigating how HPRT1 deficiency rewires nucleotide metabolism in cancer cells, potentially revealing synthetic lethal interactions or vulnerabilities exploitable in CML and other malignancies.
Typical research applications of this product include investigating purine metabolism disorders, modeling Lesch-Nyhan syndrome pathophysiology, and exploiting the HPRT1-defective background for 6-thioguanine-based counterselection in genome editing experiments. The cells can be used in enzyme activity assays, western blotting for target protein verification, RT-qPCR for expression analysis, nucleotide pool quantification, and cell viability assays with purine analogs. Additionally, they serve as a platform for high-content screening of small molecules that restore or bypass HPRT1 function, or for studying how cancer cells adapt to nucleotide salvage pathway inactivation. For further details on validation data and experimental protocols, please contact Ascent Research.
