The AMT Knockout HAP1 Polyclonal Cells are a pooled population of CRISPR/Cas9-edited HAP1 cells carrying targeted disruption of the AMT gene, which encodes the mitochondrial aminomethyltransferase enzyme. This polyclonal knockout model is designed for loss-of-function studies of AMT in a near-haploid human cell background, enabling robust and reproducible investigation of glycine metabolism and one-carbon pathway dynamics. The heterogeneous knockout population reflects a range of editing events across the AMT locus, providing a genetically diverse tool for functional genomics, drug screening, and metabolic flux analyses without the need for single-cell cloning.
HAP1 is a near-haploid human cell line derived from the KBM-7 chronic myeloid leukemia (CML) cell line. With an adherent growth morphology and a largely haploid karyotype, HAP1 cells are particularly well-suited for knockout and genetic screening applications because the presence of only one allele for most genes eliminates the complication of a second functional copy, thereby simplifying the interpretation of loss-of-function phenotypes. This host cell line has been widely adopted for CRISPR-based functional genomics due to its ease of manipulation and consistent performance in high-throughput screening formats.
AMT functions as a critical component of the mitochondrial glycine cleavage system (GCS), where it catalyzes the transfer of a methylamine group from the H protein-bound intermediate to tetrahydrofolate (THF), yielding 5,10-methylenetetrahydrofolate and free ammonia. This reaction constitutes the second step of the GCS and is tightly coupled to the activities of glycine decarboxylase (GLDC), the H protein (GCSH), and dihydrolipoamide dehydrogenase (DLD). Downstream, 5,10-methylenetetrahydrofolate serves as a key one-carbon donor for nucleotide biosynthesis, methylation reactions, and the interconversion of serine and glycine via serine hydroxymethyltransferase (SHMT1/2) and methylenetetrahydrofolate reductase (MTHFR). AMT expression and GCS flux are regulated by mitochondrial biogenesis factors such as NRF1 and TFAM, as well as by cellular glycine availability and one-carbon metabolite sensing. Loss of AMT activity disrupts glycine degradation, leading to accumulation of glycine and impaired production of one-carbon units, with profound consequences for cellular methylation capacity and ammonia detoxification.
In the HAP1 near-haploid background, disruption of AMT creates a homogeneous genetic state that facilitates the study of glycine-induced toxicity and one-carbon metabolic insufficiency. The absence of a second AMT allele eliminates compensatory gene expression, allowing direct correlation of genotype with phenotype. This model recapitulates key biochemical hallmarks of nonketotic hyperglycinemia (NKH), a severe neurometabolic disorder caused by defects in the GCS. The HAP1 AMT knockout cells thus provide a physiologically relevant platform to dissect the molecular pathogenesis of NKH and to evaluate therapeutic strategies aimed at reducing glycine levels or bypassing the metabolic block.
Researchers can employ the AMT Knockout HAP1 Polyclonal Cells for Western blotting and RT?qPCR confirmation of AMT disruption, glycine accumulation assays, 5,10-methylenetetrahydrofolate quantification, and metabolic flux analysis with labeled glycine. Viability assays under glycine stress, mitochondrial immunofluorescence, and high-content screening for glycine-lowering compounds are additional applications. These cells serve as a valuable tool for studying glycine cleavage system function, folate cycle dynamics, mitochondrial disease mechanisms, and therapeutic development for glycine encephalopathy. For further details, please contact Ascent Research.
