GBA Knockout HEK293T Cell Line

The GBA Knockout HEK293T Cell Line is a CRISPR/Cas9-edited knockout cell line that enables targeted disruption of the GBA gene, which encodes the lysosomal enzyme glucocerebrosidase (GCase). This loss-of-function model eliminates GCase activity, providing a defined human cellular platform to investigate the downstream consequences of glucosylceramide accumulation and lysosomal dysfunction. The knockout was generated in the HEK293T background using CRISPR/Cas9-mediated gene disruption, resulting in a null allele that abrogates GBA expression. This engineered cell line serves as a versatile tool for studying glycosphingolipid metabolism and its associated diseases.

HEK293T cells are a human embryonic kidney epithelial cell line stably expressing the SV40 large T antigen, which facilitates episomal replication of plasmids containing the SV40 origin of replication and enhances protein expression from transfected vectors. These cells are widely employed for transient and stable transfection, viral packaging, and high-level recombinant protein production. The epithelial morphology and high transfection efficiency make HEK293T an ideal host for generating knockout cell lines that require subsequent functional complementation or overexpression studies. In the context of lysosomal biology, HEK293T cells express key components of the endolysosomal system, enabling the analysis of GBA-dependent processes.

Glucocerebrosidase (GCase), the product of GBA, is a lysosomal hydrolase that cleaves glucosylceramide into glucose and ceramide. Its expression is regulated by TFEB and MITF, master regulators of lysosomal biogenesis. GCase activity requires interaction with Saposin C and lysosomal targeting via LIMP-2. GBA knockout abolishes GCase function, causing glucosylceramide accumulation, ceramide reduction, lysosomal pH disruption, autophagic flux impairment, and aberrant cathepsin maturation. These changes perturb the mTORC1-TFEB axis and affect pathways involving GBA2, Prosaposin, LAMP1, and Cathepsin D, ultimately driving sphingolipid imbalance and lysosomal stress.

In the HEK293T background, GBA knockout recapitulates key pathological features of Gaucher disease and Parkinson disease, including lysosomal dysfunction and impaired autophagy. The accumulation of glucosylceramide and related lipid substrates activates downstream stress responses, providing a tractable model to dissect disease mechanisms. This cell line allows precise control over experimental conditions, enabling detailed biochemical and cell biological analyses of lysosomal storage disorders. It is particularly suited for rescue experiments with wild-type or mutant GBA variants to validate genotype-phenotype relationships, as well as for mechanistic studies linking GBA deficiency to ??-synuclein accumulation and neurodegeneration.

This cell line supports diverse research applications, such as drug screening for pharmacological chaperones, autophagy assessment via LC3 turnover, and lipid quantification by LC-MS. LysoTracker staining, LAMP1 immunofluorescence, and RT-qPCR for TFEB target genes facilitate functional dissection of lysosomal dysfunction. Western blotting and enzyme activity assays confirm GBA disruption. The model is also amenable to genome-wide CRISPR screens for modifiers of glucosylceramide toxicity. For further information or to request a quote, please contact Ascent Research.