The AGPAT4 Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal cell population derived from the HEK293T human embryonic kidney epithelial line, engineered for targeted disruption of the AGPAT4 gene. This polyclonal knockout model comprises a heterogeneous pool of cells carrying diverse loss-of-function alleles introduced at the AGPAT4 locus, enabling robust study of gene ablation effects without clonal selection. The pooled format preserves population-level genetic variation, making it suitable for experiments requiring physiological relevance and avoiding artifacts associated with single-cell cloning. Researchers can use these cells to interrogate AGPAT4-dependent pathways in a well-characterized host background amenable to high-throughput screening and detailed molecular analysis.
HEK293T cells are a widely adopted derivative of the HEK293 cell line, distinguished by stable expression of the SV40 large T antigen. This modification permits high-level episomal replication of vectors containing the SV40 origin of replication, resulting in transient protein overexpression at levels unattainable in standard HEK293 cells. The cells display epithelial morphology, adherent growth, and exceptional transfectability, making them a workhorse for gene function studies, protein?Cprotein interaction assays, and signaling pathway dissection. Their human origin ensures that cellular machinery and regulatory networks are physiologically relevant for modeling human diseases, particularly in the contexts of metabolic and proliferative signaling.
AGPAT4 (1-acylglycerol-3-phosphate O-acyltransferase 4) encodes a key enzyme in lipid biosynthesis that catalyzes the conversion of lysophosphatidic acid (LPA) to phosphatidic acid (PA) using acyl-CoA as a co-substrate. This reaction lies at a critical branch point in glycerophospholipid metabolism and triacylglycerol synthesis, placing AGPAT4 upstream of lipin-mediated diacylglycerol generation and downstream of glycerol-3-phosphate acyltransferases (GPATs). PA itself functions as a signaling lipid that directly activates mTORC1, leading to phosphorylation of downstream effectors S6K and 4E-BP1 and thereby integrating nutrient-sensing and cell growth regulation. AGPAT4 expression is transcriptionally regulated by SREBP1c and PPAR??, and its activity is modulated by insulin/IGF-1 signaling. Additionally, AGPAT4 interacts with protein kinase D and the mTOR complex, situating it within a network that couples membrane phospholipid production to anabolic responses.
In the HEK293T background, disruption of AGPAT4 provides a powerful tool for dissecting the intersection of lipid metabolism and cellular growth control. These cells constitutively exhibit active insulin receptor/IGF-1 receptor signaling and robust translational machinery, making them acutely sensitive to alterations in mTORC1 activity. Loss of AGPAT4 is expected to reduce cellular PA pools, thereby attenuating mTORC1-dependent phosphorylation of S6K and 4E-BP1 and potentially impacting proliferation and global protein synthesis. Furthermore, because HEK293T cells are not specialized for lipid storage, the model facilitates the study of general metabolic reprogramming and PA-mediated signaling without confounding differentiation-related lipid droplet accumulation. Researchers can reconstitute the pathway by overexpressing wild-type or mutant AGPAT4 to validate functional consequences, thanks to the high transgene expression achievable in these cells.
This knockout cell population is engineered for a broad array of mechanistic and translational applications. Key uses include probing glycerophospholipid flux via lipidomic LC-MS profiling, quantifying PA levels with enzymatic or mass-based assays, and monitoring mTORC1 activation status through phospho-S6K Western blotting. The model is suitable for investigating insulin resistance mechanisms, testing small-molecule inhibitors targeting lipid metabolic enzymes, and validating AGPAT4 as a candidate drug target in metabolic syndrome and type 2 diabetes. Additional experimental designs can incorporate lipid accumulation staining after ectopic expression of adipogenic transcription factors to model aspects of obesity-related fat storage. These cells also support studies on phospholipase D signaling crosstalk and PA-binding kinase cascades. For further information or to discuss customized applications, please contact Ascent Research.
