The HEATR5B Knockout HEK293T Polyclonal Cells product provides a versatile CRISPR/Cas9-edited polyclonal knockout cell population targeted for disruption of the HEATR5B gene in the human embryonic kidney HEK293T host cell line. This polyclonal pool is generated through CRISPR/Cas9-mediated gene disruption, producing a heterogeneous population of cells with loss-of-function mutations in HEATR5B. The resulting model offers a robust system for investigating the role of HEATR5B in lysosomal biology, mTORC1 signaling, and autophagy, without the need for single-cell clone isolation. The polyclonal format retains genetic diversity while enabling consistent functional knockout across the population, making it suitable for both pathway analysis and functional screening applications.
The host cell line, HEK293T, is an adherent, epithelial cell line derived from human embryonic kidney cells and stably expresses the SV40 large T antigen. This modification enhances episomal replication of plasmids containing the SV40 origin, leading to high transfection efficiency and robust protein expression. HEK293T cells are widely employed as a versatile workhorse in biomedical research for applications including transient and stable protein production, lentivirus and retrovirus package, and gene editing experiments. Their fast growth rate and well-characterized genetic background make them an ideal chassis for generating knockout models to dissect complex signaling pathways.
HEATR5B (HEAT Repeat Containing 5B) functions as a critical lysosomal scaffold protein that coordinates lysosomal positioning and amino acid-dependent mTORC1 signaling. Mechanistically, HEATR5B is activated by amino acid stimulation and contributes to the recruitment of mTORC1 to the lysosomal surface through direct interactions with the Rag GTPases (RagA and RagC), the Ragulator complex (LAMTOR1-5), and v-ATPase subunits. Upon activation, mTORC1 phosphorylates downstream targets including S6K at Thr389 and 4EBP1, driving protein synthesis and inhibiting autophagy. Conversely, HEATR5B loss uncouples mTORC1 from lysosomes, leading to reduced phosphorylation of S6K and 4EBP1, and de-repression of autophagy through TFEB and ULK1. This scaffolding protein thus integrates nutrient signals from upstream regulators (including amino acids, insulin/IGF-1, and AMPK) to balance anabolic and catabolic processes.
In the HEK293T cellular context, disruption of HEATR5B creates a valuable model to explore lysosomal biology and metabolic regulation. Because HEK293T cells have high secretory and biosynthetic capacity and rely on robust mTORC1 activity for growth, HEATR5B knockout provides a clean genetic background to assess consequences of impaired lysosomal positioning and mTORC1 signaling. Researchers can directly examine the interdependency between lysosomal trafficking, mTORC1 kinase activity, and autophagic flux. This model is especially useful for studying how lysosomal scaffold dysfunction influences cancer-relevant metabolic rewiring, as HEK293T cells exhibit many transformed characteristics.
Research applications for this polyclonal knockout product encompass a broad spectrum of assays aimed at dissecting mTORC1 signaling, lysosomal dynamics, and autophagy. Typical experiments include western blotting to detect phospho-S6K (T389) and phospho-4EBP1 changes under nutrient-starvation and refeeding conditions, immunofluorescence analysis of mTOR and LAMP2 colocalization, LysoTracker staining for lysosomal mass and positioning, and autophagy flux measurement via LC3 lipidation and p62 degradation. Co-immunoprecipitation studies can validate disrupted interactions between HEATR5B and Rag GTPases. Furthermore, these cells are suitable for functional screens to identify modulators of lysosomal function or mTORC1 activity. For additional technical details or support, please contact Ascent Research.
