The GPAT4 Knockout HAP1 Polyclonal Cells constitute a CRISPR/Cas9-edited polyclonal knockout cell population in which the human GPAT4 gene has been disrupted, generating a loss-of-function model for investigating glycerolipid biosynthesis. This polyclonal format provides a heterogeneous mixture of edited cells, facilitating robust population-level analyses of GPAT4-dependent phenotypes without the constraints of clonal selection. The knockout model is designed for functional genomics, metabolic studies, and drug discovery applications.
The host HAP1 cell line is a human near-haploid chronic myeloid leukemia (CML) line derived from the male KBM-7 cell line. HAP1 cells exhibit an adherent, fibroblast-like morphology and retain a haploid karyotype for most chromosomes, enabling efficient CRISPR/Cas9-mediated gene targeting and straightforward genotype?Cphenotype correlation. This genetic simplicity makes HAP1 an ideal platform for studying gene function in a clean, diminished background, particularly for genes involved in fundamental cellular processes such as lipid metabolism.
GPAT4 encodes a mitochondrial glycerol-3-phosphate acyltransferase that catalyzes the initial acylation of glycerol-3-phosphate to form lysophosphatidic acid (LPA), a pivotal intermediate in glycerolipid and phospholipid synthesis. GPAT4 activity is tightly regulated by adipogenic transcription factors PPAR?? and SREBP-1c, as well as insulin signaling and ChREBP, placing it at the nexus of energy sensing and lipid anabolism. The resulting LPA is further acylated by AGPAT enzymes to generate phosphatidic acid (PA), which is then converted by LPIN1 (lipin) to diacylglycerol (DAG). DAG serves as a substrate for triacylglycerol (TAG) synthesis via DGAT enzymes, ultimately leading to lipid droplet biogenesis and storage. GPAT4 also interfaces with lipid droplet-associated proteins such as Seipin and Perilipin-2, underscoring its functional integration within the lipid droplet machinery.
In the HAP1 background, disruption of GPAT4 results in a marked reduction in de novo glycerolipid synthesis, providing a clean cellular system to dissect the contribution of this specific acyltransferase isoform to neutral lipid accumulation. The leukemic origin of HAP1 cells offers a unique context to explore cancer-relevant lipid dysregulation, as aberrant lipid metabolism is increasingly recognized in leukemia propagation and drug resistance. Furthermore, the haploid genome reduces genetic redundancy, allowing clear attribution of metabolic defects to GPAT4 loss and enabling high-content screening for modulators of lipid storage pathways.
This knockout model supports a broad array of experimental approaches, including Oil Red O and BODIPY staining for lipid droplet visualization, triglyceride quantification assays, fatty acid uptake measurements, and adipogenic differentiation protocols coupled with PPAR?? reporter assays. Transcriptomic profiling via RNA-seq can reveal global changes in lipid metabolism gene expression upon GPAT4 ablation. The cells are well-suited for functional validation of candidate genes in congenital generalized lipodystrophy, obesity, and hepatic steatosis, as well as for small-molecule screening aimed at restoring or bypassing defective TAG synthesis. For additional details or custom inquiries, please contact Ascent Research.
