The AMPD3 Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population in which the AMPD3 gene has been disrupted in the HAP1 human near-haploid cell line. This model serves as a loss-of-function tool for studying AMPD3??s role without requiring single-cell cloning, offering a heterogeneous yet representative knockout background. The polyclonal format is ideal for high-throughput screening and bulk functional assays, leveraging HAP1??s haploid genetic simplicity.
HAP1 is a fibroblast-like suspension cell line derived from the KBM-7 chronic myeloid leukemia line and maintains a near-haploid karyotype. This unique characteristic simplifies gene-function analysis, as virtually all genes are present in a single copy, reducing genetic redundancy and enhancing the efficiency of knockout screening. HAP1 cells are widely used in functional genomics, haploid genetic screens, and drug discovery due to their rapid proliferation and compatibility with multiwell assays, flow cytometry, and metabolite profiling.
The AMPD3 gene encodes adenosine monophosphate deaminase 3, which catalyzes the hydrolytic deamination of AMP to IMP, a key step in the purine nucleotide cycle. This reaction is central to adenine nucleotide homeostasis and connects amino acid catabolism to energy metabolism. AMPD3 activity is modulated by the AMP/ATP ratio and is transcriptionally regulated by MYC and HIF1A, placing it downstream of nutrient-sensing and hypoxic signaling. The enzyme assembles as a homotetramer and functionally interacts with adenylosuccinate synthetase (ADSS), adenylosuccinate lyase (ADSL), and IMP dehydrogenase (IMPDH) within the cycle. Its reaction generates IMP and fumarate, which contribute to GTP and ATP pools, and influences aspartate utilization. Knockout of AMPD3 disrupts ATP homeostasis and fumarate production, leading to imbalanced nucleotide pools that are implicated in erythrocyte AMP deaminase deficiency, neurodevelopmental disorders with spasticity, and aberrant cancer metabolism.
In the HAP1 cellular context, the near-haploid background and dependence on high energy turnover make AMPD3 disruption a powerful model for studying metabolic vulnerabilities and nucleotide pool regulation. The polyclonal nature avoids clonal artifacts and permits robust assessment of population-level metabolic shifts, such as altered AMP/ATP ratios or IMP levels, using HPLC and ATP/ADP/AMP ratio assays. Its suspension growth format enables scalable screening for phenotypic changes via flow cytometry and cell viability readouts, and facilitates metabolomic sampling. This system is especially suited for investigating how AMPD3 loss affects the purine nucleotide cycle under stress conditions or oncogenic signaling.
This AMPD3 knockout population is applicable across a range of research areas. It supports functional dissection of purine metabolism, analysis of nucleotide salvage pathways, and modeling of neurodevelopmental disorders associated with AMPD3 mutations. In cancer biology, it can be used to explore metabolic dependencies and validate AMPD3 as a drug target. Common downstream assays include Western blotting for AMPD3 and pathway markers, RT-qPCR for transcript verification, HPLC-based nucleotide quantification, AMP deaminase enzymatic activity measurement, and cell viability or proliferation tests. The polyclonal format also facilitates pooled CRISPR screens and combinatorial drug studies. For further information, please contact Ascent Research.
