The ATXN3 knockout HEK293T polyclonal cells are a CRISPR/Cas9-edited polyclonal population in which the ATXN3 gene has been disrupted to create a loss-of-function model. This heterogeneous knockout pool, generated through CRISPR/Cas9-mediated gene disruption, avoids the limitations of single-cell cloning and allows analysis of ataxin-3 function across a genetically diverse cellular background.
The host cell line, HEK293T, is a human embryonic kidney epithelial derivative that stably expresses the SV40 large T antigen, enabling high-copy episomal replication of plasmids and robust recombinant protein expression. Widely adopted for viral production and signal transduction studies, HEK293T cells provide an experimentally tractable system for examining protein quality control, the ubiquitin-proteasome system, and autophagy due to their active secretory pathway and well-characterized stress response machinery.
ATXN3 encodes the deubiquitinase ataxin-3, which removes ubiquitin chains from substrate proteins to modulate proteasomal degradation and autophagic clearance. Ataxin-3 functions downstream of the stress-inducible transcription factor HSF1 and forms complexes with VCP/p97, ubiquilin-1, and the co-chaperone CHIP, along with HSP70 and HSP90, to mediate recognition and processing of misfolded proteins. This enzyme regulates the stability of p53 and PTEN and directs aggregated proteins to aggresomes, thereby coordinating responses to proteotoxic stress. Under disrupted proteostasis, ATXN3 also influences autophagic flux, as indicated by changes in LC3 lipidation and p62 degradation.
In HEK293T cells, ATXN3 knockout disrupts protein quality control, leading to accumulation of polyubiquitinated species and impaired degradation of aggregation-prone substrates. The loss of ataxin-3 function compromises aggresome formation and autophagic turnover, sensitizing cells to endoplasmic reticulum stress and elevating markers such as BiP and CHOP. These cellular phenotypes mimic aspects of Machado-Joseph disease pathology, making the model valuable for studying spinocerebellar ataxia type 3 and other polyglutamine disorders.
Typical applications include western blot analysis of ubiquitin conjugates, proteasome activity assays using fluorogenic substrates, autophagy flux measurements by monitoring LC3-II turnover, and immunofluorescence microscopy for aggresome detection. This polyclonal knockout product supports drug screening for modulators of protein aggregation and deubiquitinase activity, as well as functional complementation studies with ATXN3 variants. Researchers can also investigate crosstalk between the proteasome and autophagy under basal and stressed conditions. For further technical inquiries, please contact Ascent Research.
