ALDH4A1 Knockout HAP1 Polyclonal Cells provide a CRISPR/Cas9-edited polyclonal knockout population in the HAP1 cell line, with targeted disruption of the ALDH4A1 gene. This pooled population contains a variety of loss-of-function alleles generated without clonal selection, enabling efficient functional analysis of ALDH4A1 in a near-haploid genetic setting. The polyclonal format preserves cellular heterogeneity and facilitates robust phenotypic screening, making it suitable for studies in proline metabolism and redox biology.
The HAP1 cell line is a near-haploid derivative of the KBM-7 chronic myeloid leukemia (CML) line, isolated from a male patient. Its haploid karyotype simplifies genome editing, as a single targeted disruption can produce a functional null phenotype, avoiding the complexity of diploid alleles. HAP1 is widely adopted for genetic knockout studies, haploid screens, and drug mechanism investigations, and its CML origin adds relevance for hematological cancer research.
ALDH4A1 is a mitochondrial dehydrogenase that catalyzes the NAD+-dependent conversion of P5C to glutamate, a rate-limiting step in proline catabolism. Proline is first oxidized by PRODH to P5C, which ALDH4A1 further metabolizes to glutamate, feeding the TCA cycle via ??-ketoglutarate and supporting ATP production. This pathway also mitigates ROS from proline oxidation and supplies glutamate for glutathione synthesis, maintaining redox balance. ALDH4A1 expression is regulated by TP53, proline levels, oxidative stress, and PI3K/AKT signaling. Its activity impacts downstream glutamate availability, glutathione production, and anaplerotic replenishment of TCA cycle intermediates, underscoring its role in metabolic and stress integration.
In HAP1 cells, ALDH4A1 knockout disrupts the connection between proline degradation and core metabolism, offering a powerful model to study metabolic reprogramming in cancer. Cancer cells often rely on proline metabolism for energy and redox control, and loss of ALDH4A1 can reveal vulnerabilities in glutamate and glutathione pathways under nutrient stress. The near-haploid background ensures high penetrance of the knockout phenotype without compensatory alleles. Additionally, with TP53 governing upstream PRODH, this model is instrumental for dissecting p53-mediated metabolic responses to oxidative stress, enabling mechanistic studies of stress-induced cell death and potential therapeutic targets.
Applications include functional genomics, cancer metabolism analysis, hyperprolinemia type II modeling, and drug target validation. Compatible assays range from P5C/proline quantification and glutamate measurement to TCA cycle metabolite profiling via mass spectrometry. Western blotting and RT-qPCR confirm ALDH4A1 loss, while ROS detection and viability assays evaluate functional consequences. Immunofluorescence can probe mitochondrial integrity. For further technical support or custom inquiries, please contact Ascent Research.
