The GLA Knockout Raji Polyclonal Cells are a CRISPR/Cas9-edited polyclonal population of Raji B lymphoblasts with targeted disruption of the GLA gene encoding lysosomal ??-galactosidase A. This gene disruption establishes a loss-of-function model that eliminates ??-galactosidase A enzymatic activity, serving as a system to dissect Fabry disease pathobiology and glycosphingolipid metabolism in a B lymphocyte cellular context. The polyclonal format provides a heterogeneous knockout pool that mitigates clonal selection biases and is well-suited for population-level functional analyses.
The Raji host cell line is an Epstein-Barr virus (EBV)-positive Burkitt lymphoma-derived B lymphocyte line. Raji cells are extensively characterized for their robust B cell receptor signaling, antigen presentation machinery, and constitutive immunoglobulin production. As a suspension-adapted line with defined culture requirements, Raji offers a reliable and tractable cellular background for CRISPR-based gene disruption and subsequent investigation of B cell-related pathways.
The GLA gene product, ??-galactosidase A, is a lysosomal hydrolase responsible for cleaving terminal ??-galactose residues from globotriaosylceramide (Gb3) and other glycoconjugates. In the lysosome, the enzyme acts in conjunction with the sphingolipid activator protein Saposin B and the membrane protein LAMP-2, with proper folding assisted by HSP70 molecular chaperones. Disruption of GLA blocks Gb3 catabolism, leading to intracellular accumulation of Gb3, its deacylated form lyso-Gb3, and upstream sphingolipid intermediates such as ceramide and glucosylceramide. Gb3 itself is synthesized by Gb3 synthase (A4GALT) from lactosylceramide, which is generated through the sequential actions of glucosylceramide synthase (UGCG) and lactosylceramide synthase. Upstream transcriptional regulators including TFEB and MITF control lysosomal biogenesis and GLA expression, while androgens and inflammatory cytokines can modulate ??-galactosidase A levels, embedding the knockout in a complex regulatory network.
In the Raji B cell background, GLA deficiency leads to lysosomal dysfunction, impaired autophagic flux, and cellular stress driven by Gb3 storage??pathological features that closely mimic Fabry disease, an X-linked lysosomal storage disorder. This model uniquely enables the study of how glycosphingolipid accumulation impacts B lymphocyte functions, such as antigen presentation, cytokine secretion, and B cell receptor signaling, which may contribute to the immune dysregulation reported in Fabry patients. Additionally, the EBV-immortalized, lymphoma-derived nature of Raji cells allows exploration of the convergence between oncogenic signaling and lysosomal stress responses.
Key research applications include modeling Fabry disease pathophysiology, assessing enzyme replacement therapies with recombinant ??-galactosidase A, and screening pharmacological chaperones to stabilize misfolded enzyme variants. The cells also support broader studies of sphingolipid metabolism and lysosomal biology. Common experimental readouts include Western blotting for GLA protein, ??-galactosidase A enzymatic activity assays, immunofluorescence detection of Gb3 deposits, Lysotracker staining for lysosomal mass/pH, LC-MS/MS lipidomics to quantify Gb3 and lyso-Gb3, autophagy flux monitoring via LC3 turnover, RT-qPCR profiling of lysosomal and stress-responsive genes, and flow cytometric analysis of immune surface markers. For further technical information, please contact Ascent Research.
