ATXN3 Knockout HeLa Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from the HeLa cell line, designed for loss-of-function studies of the ATXN3 gene. The knockout is achieved through CRISPR/Cas9-mediated gene disruption, creating a heterogeneous pool of cells with targeted inactivation of ATXN3. This polyclonal format provides a robust model system for investigating the cellular functions of ataxin-3 without the clonal selection biases, suitable for population-based assays.
HeLa cells are an immortalized epithelial cell line originally derived from a cervical adenocarcinoma of a 31-year-old African American woman. They harbor human papillomavirus type 18 (HPV18) sequences and display a hypertriploid karyotype, growing as an adherent monolayer. HeLa cells are widely utilized in biomedical research as a versatile model for human cell biology, including studies of cancer mechanisms, protein homeostasis, and cellular stress responses. Their robust growth and ease of manipulation make them an ideal host for generating knockout models to dissect gene function.
ATXN3 encodes ataxin-3, a deubiquitinating enzyme that edits polyubiquitin chains on substrate proteins, primarily through its Josephin domain. Ataxin-3 functions in the ubiquitin-proteasome system by interacting with the VCP/p97 complex and proteasome subunits, facilitating the extraction and degradation of misfolded or ubiquitinated proteins. It is regulated by heat shock factor 1 (HSF1) under proteotoxic stress and participates in the clearance of aggregate-prone proteins via aggresome formation and autophagy. Key molecular partners include HSP70, HSP90, CHIP, and p62/SQSTM1. Downstream targets encompass a broad array of ubiquitinated proteins and the tumor suppressor p53. Disruption of ATXN3 impairs deubiquitinase activity and disrupts protein quality control networks, leading to accumulation of polyubiquitinated species and altered proteasomal degradation.
In the context of HeLa cells, ATXN3 knockout provides a powerful tool to examine the role of ataxin-3 in protein homeostasis independently of the polyglutamine expansions that underlie Machado-Joseph disease. This model allows researchers to assess how loss of ATXN3 affects ubiquitin-dependent pathways, proteasome activity, and cellular responses to proteotoxic insults such as proteasome inhibition or oxidative stress. Since HeLa cells are proficient in aggregation-prone protein handling when artificially expressed, the knockout background enables precise evaluation of ataxin-3??s contribution to aggresome formation and autophagy-mediated clearance. Moreover, the polyclonal nature avoids artifacts from individual clonal variants, reflecting a physiological range of knockout effects.
This knockout cell population supports a wide array of experimental applications, including deubiquitinase enzymology assays, western blotting for ubiquitinated proteins, immunofluorescence for protein aggregates, and filter trap assays to monitor aggregation. It is suitable for investigating proteasome and autophagy regulation, as well as high-throughput screening for modifiers of protein aggregation relevant to Machado-Joseph disease and other polyglutamine disorders. Co-immunoprecipitation and proteasome activity assays can be employed to map interactomes and functional consequences. For further technical specifications, validation data, or ordering information, please contact Ascent Research.
