This product is a CRISPR/Cas9-edited polyclonal knockout cell population targeting the human AP3D1 gene in HEK293T cells. The polyclonal format represents a heterogeneous pool of edited cells following CRISPR/Cas9-mediated gene disruption, suitable for analyzing loss-of-function effects without clonal selection. This knockout model abrogates expression of the delta subunit of the adaptor protein complex AP-3, a critical component of clathrin-dependent trafficking from the trans-Golgi network (TGN) to lysosomes and lysosome-related organelles.
The HEK293T host cell line is a derivative of the human embryonic kidney HEK293 line, stably expressing the SV40 large T-antigen. This modification enables high-level episomal replication of plasmids containing the SV40 origin, making HEK293T a widely used host for transient protein expression, lentivirus and retrovirus production, and functional genomics studies. The epithelial origin and robust transfectability of HEK293T provide an experimentally tractable system for investigating intracellular membrane trafficking and organelle biogenesis.
AP3D1 encodes the delta subunit of the heterotetrameric AP-3 adaptor complex, which sorts transmembrane cargo from the TGN into clathrin-coated vesicles destined for lysosomes, melanosomes, and platelet dense granules. The AP-3 complex binds tyrosine-based motifs in cargo such as LAMP1, LAMP2, and tyrosinase, a process regulated by ARF-family GTPases and phosphatidylinositol 3-kinase. It interacts with clathrin and adaptin subunits AP3B1, AP3M1/2, and AP3S1/2 to drive vesicle formation. Proper AP-3 function ensures delivery of lysosomal membrane proteins for organelle acidification, melanogenic enzymes TYR and TYRP1 to melanosomes, and bioactive molecules like serotonin to platelet dense granules. AP3D1 disruption thus causes cargo mislocalization and organelle dysfunction.
In the HEK293T background, knockout of AP3D1 replicates key cellular phenotypes associated with Hermansky-Pudlak syndrome type 10 (HPS10), a disorder characterized by oculocutaneous albinism, platelet storage pool deficiency, and immune dysfunction. Although HEK293T cells do not form melanosomes or platelet dense granules, they possess a robust lysosomal system, making them suitable for studying lysosomal biogenesis and the trafficking of integral membrane proteins. AP3D1 disruption leads to accumulation of LAMP1 at the plasma membrane and defective lysosomal targeting, as well as altered autophagic flux, providing a simplified model for investigating AP-3-dependent sorting mechanisms.
Researchers can employ this polyclonal knockout pool in diverse experimental workflows, including immunofluorescence microscopy for LAMP1 mislocalization, western blotting and flow cytometry to monitor lysosomal protein trafficking, and RT-qPCR for transcriptional profiling. The polyclonal format is valuable for pooled screening and generating populations for downstream clonal isolation. This product is a versatile tool for studying clathrin-mediated sorting, modeling Hermansky-Pudlak syndrome, and screening compounds that restore lysosomal function. For further inquiries, contact Ascent Research.
