HTT Knockout HEK293T Polyclonal Cells are a population of HEK293T cells modified by CRISPR/Cas9 to disrupt the HTT gene, which encodes the huntingtin protein. This polyclonal knockout model provides a genetically heterogeneous pool, enabling robust analysis of HTT-dependent phenotypes without the selective effects of clonal expansion. Loss of huntingtin expression can be confirmed by anti-huntingtin Western blot, making these cells suitable for investigating consequences of huntingtin deficiency in a non-neuronal context.
The host cell line, HEK293T, is an immortalized human embryonic kidney epithelial derivative expressing SV40 large T antigen. This background confers high transfection efficiency and supports high-level recombinant protein expression, making it a workhorse for cell biology and biochemistry assays. Although non-neuronal, HEK293T cells express core trafficking and signaling machinery, allowing dissection of huntingtin’s fundamental roles independent of neuronal specialization.
Huntingtin functions as a scaffold protein interacting with HAP1, dynein, and kinesin to mediate vesicle trafficking, including transport of BDNF-containing vesicles. It also regulates autophagy and apoptosis. Upstream, AKT and CDK5 phosphorylate huntingtin, while caspase-3 and calpain cleave it. Downstream, huntingtin modulates REST/NRSF-mediated gene repression and promotes PGC-1??-dependent mitochondrial biogenesis. In BDNF signaling, huntingtin couples TrkB activation to retrograde transport of trophic signals, essential for neuronal survival.
In HEK293T cells, HTT disruption results in deficient huntingtin, leading to impaired autophagy flux, disrupted BDNF secretion, and aberrant trafficking. This model allows direct examination of these processes without neuronal confounding factors, ideal for modifier screens and drug discovery. Assays such as autophagy flux analysis with lysosomal inhibitors, BDNF ELISA, and immunofluorescence for huntingtin interactors (e.g., HAP1) can be readily performed. The polyclonal nature averages out stochastic effects, providing a stable population for repeated experiments.
Applications include Huntington disease modeling, high-throughput screening for HTT pathway modulators, and mechanistic studies of BDNF/TrkB signaling and endosomal trafficking. Standard characterization assays encompass anti-huntingtin Western blot, RT-qPCR, immunofluorescence, autophagy flux measurement, BDNF ELISA, and MTT viability assays. The cells also support live-cell imaging of fluorescently tagged trafficking markers. For additional information or technical support, please contact Ascent Research.
