This product is a polyclonal knockout cell population generated by CRISPR/Cas9-mediated disruption of the ALDH18A1 gene in the HAP1 cell line. The knockout pool contains a heterogeneous mixture of edited alleles, offering a functional loss-of-function model without clonal selection.
The HAP1 line is a human near-haploid cell model derived from the KBM-7 chronic myeloid leukemia parental line. Its near-haploid karyotype simplifies genetic analysis and enables efficient homozygous knockout upon single-allele targeting, making it a preferred host for genetic screens, knockout validation, and cancer research.
The ALDH18A1 gene product, pyrroline-5-carboxylate synthase (P5CS), is a mitochondrial bifunctional enzyme catalyzing the first two steps of de novo proline biosynthesis. It converts glutamate to ??-glutamyl phosphate via its N-terminal kinase domain and then reduces the intermediate to glutamic-??-semialdehyde via its C-terminal reductase domain; the semialdehyde cyclizes to P5C. P5C is a metabolic branch point: reduction to proline by P5C reductase or conversion to ornithine. The pathway is linked to the urea cycle and arginine metabolism. Transcription of ALDH18A1 is under control of c-MYC and ATF4, tying proline synthesis to cellular proliferation and stress. Proline is critical for collagen stability, and its deficiency disrupts extracellular matrix integrity. Knockout of ALDH18A1 eliminates P5CS activity, inducing proline auxotrophy and potentially impairing arginine synthesis and collagen production.
Within the HAP1 near-haploid system, ALDH18A1 knockout circumvents complexities of diploid compensation, providing a robust platform for studying metabolic pathway flux and genetic interactions. The resulting proline dependency can be exploited to probe amino acid sensing, mTOR signaling, and redox homeostasis. Furthermore, the model offers a tractable system to investigate disease mechanisms of autosomal recessive cutis laxa type IIIA and spastic paraplegia type 9B, both associated with deleterious ALDH18A1 mutations.
Common research uses include proline and arginine metabolism studies, cutis laxa and neurodevelopmental disorder modeling, cancer metabolism investigation, and haploid genetic interaction screens. Experimentally, researchers can assess knockout efficiency via RT-qPCR and anti-P5CS Western blot, measure intracellular proline using HPLC or colorimetric assays, evaluate cell viability under proline withdrawal, determine collagen synthesis by [3H]-proline incorporation or hydroxyproline assay, and perform cell cycle analysis by flow cytometry. The polyclonal population is well suited for pooled modifier screens and metabolic drug testing. Contact Ascent Research for additional product information and support.
