The BCAT1 Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population that provides targeted disruption of the BCAT1 gene in a haploid human cell background. This mixed pool of edited HAP1 cells constitutes a loss-of-function model for investigating BCAT1 without clonal isolation, reducing clonal biases and preserving genetic variability suited for pooled functional genomics studies.
HAP1 is a near-haploid cell line derived from KBM-7 chronic myeloid leukemia (CML) blast crisis cells, containing a single chromosome set except for a disomic region on chromosome 15. The haploid karyotype facilitates efficient CRISPR/Cas9-mediated gene knockout, as a single-allele disruption yields a null phenotype, enabling unambiguous genotype-phenotype interpretation. Widely adopted for functional genomics and drug screening, HAP1 cells maintain key myeloid features, making them relevant for modeling hematologic malignancies and BCAA metabolism.
BCAT1 encodes a pyridoxal phosphate-dependent cytosolic aminotransferase that catalyzes transamination of leucine, isoleucine, and valine to produce glutamate and branched-chain ??-keto acids. This enzymatic step links BCAA catabolism to mTORC1 activation via ??-ketoisocaproate and to nucleotide biosynthesis through glutamate-dependent nitrogen transfer to CAD and DHODH. BCAT1 is transcriptionally regulated by HIF-1?? and c-Myc, and functions upstream of mTORC1 and glutamate dehydrogenase, while interacting with BCAT2. Consequently, BCAT1 integrates amino acid metabolism with anabolic signaling and redox homeostasis.
Because HAP1 cells originate from CML blast crisis, BCAT1 knockout in this context directly addresses its role in myeloid malignancy. Disruption of BCAT1 eliminates the enzyme’s contributions to aberrant mTORC1 activity and nucleotide biosynthesis, providing a clean isogenic background to test dependencies on BCAT1-mediated metabolic rewiring. The haploid nature ensures all polyclonal cells carry the knockout allele, enabling robust bulk analyses of proliferation, apoptosis, and drug responses without wild-type interference.
This product supports diverse applications, including 13C-leucine isotopic tracing to map BCAA metabolic fluxes, Western blotting for BCAT1 and mTORC1 pathway effectors (phospho-S6K1, total S6K1), and RT-qPCR for BCAT1 transcript. Functional assays such as CellTiter-Glo viability, Annexin V/7-AAD apoptosis, and cell cycle flow cytometry quantify growth phenotypes. Drug sensitivity profiling with BCAT1 inhibitors can uncover synthetic vulnerabilities. The polyclonal pool is also suitable for genome-scale CRISPR screens. For additional information, contact Ascent Research.
