The CD320 Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal cell population derived from the HAP1 cell line, with targeted disruption of the CD320 gene encoding the transcobalamin receptor. This polyclonal format provides a heterogeneous pool of knockout cells that collectively establish a loss-of-function model suitable for functional studies without clonal isolation. The population is unselected, reflecting a spectrum of edits that broadly ablate receptor activity, enabling robust phenotypic assessments in bulk culture.
HAP1 is a human near-haploid cell line originally derived from the KBM-7 chronic myeloid leukemia line. It retains a near-haploid karyotype, with disomy for chromosome 8, which simplifies genome editing and reduces allelic background effects. This feature has made HAP1 a favored model for CRISPR/Cas9-based functional genomics and knockout screens, particularly for studying genes involved in metabolism and signaling where gene dosage effects are minimized.
CD320 encodes the transcobalamin receptor that mediates cellular uptake of the transcobalamin (TCN2)-cobalamin (vitamin B12) complex via receptor-mediated endocytosis, facilitated by LRP2. Transcriptional regulation involves SP1 and NF-Y transcription factors. Internalized cobalamin is processed in lysosomes to generate methylcobalamin and adenosylcobalamin, cofactors for methionine synthase (MTR) and methylmalonyl-CoA mutase (MMUT), respectively. This positions CD320 at a critical node linking cobalamin transport to homocysteine remethylation and methylmalonic acid catabolism, with direct consequences for one-carbon metabolism and mitochondrial propionate handling.
In the HAP1 near-haploid background, CD320 knockout recapitulates a severe block in cobalamin acquisition, providing a defined model for transcobalamin receptor deficiency. The polyclonal population mirrors heterogeneous lesion types, enabling study of phenotypic variability and threshold effects in B12-dependent pathways. This model is pertinent to research on methylmalonic aciduria, megaloblastic anemia, and the crosstalk between vitamin B12 status and methionine?cycle intermediates, with heightened phenotypic clarity due to haploid genetics.
The knockout cells support diverse experimental applications, including [57Co]-cobalamin uptake assays, intracellular cobalamin quantification by mass spectrometry, methylmalonic acid (MMA) and homocysteine measurements, and enzymatic activity assays for MTR and MMUT. Techniques such as western blotting, RT?qPCR for CD320, and cell proliferation under low?B12 conditions permit comprehensive characterization. The model is amenable to drug screening for B12?related disorders. For further technical information, please contact Ascent Research.
