This product comprises a CRISPR/Cas9-edited polyclonal knockout cell population derived from HEK293T cells, engineered for targeted disruption of the B3GNT5 gene. These polyclonal knockout cells offer a heterogeneous loss-of-function model, ideal for studying the collective impact of B3GNT5 ablation on glycosphingolipid biosynthesis without selecting for clonal isolates. The knockout model is generated using non-viral, transient delivery of Cas9 ribonucleoprotein complexes and guide RNAs, resulting in a mixed population of edited alleles that collectively eliminate functional B3GNT5 expression. This approach avoids the artifacts and selective pressures associated with clonal expansion, providing a robust platform for investigating glycan-dependent cellular processes in a physiologically relevant context.
The host cell line, HEK293T, is a female human embryonic kidney epithelial line transformed with adenovirus type 5 DNA and stably expressing the SV40 large T antigen. This enables high-copy episomal replication of plasmids containing the SV40 origin of replication, making HEK293T the workhorse for transient protein expression and viral packaging. In the knockout context, the HEK293T background provides a well-characterized glycan biosynthesis machinery, with endogenous expression of relevant glycosyltransferases and substrates. Its robust growth and ease of transfection facilitate downstream functional assays, including glycomic profiling and cell adhesion studies. The cell line??s epithelial origin also makes it a relevant model for exploring glycosphingolipid roles in epithelial cell behavior and transformation.
B3GNT5 encodes a UDP-GlcNAc:beta-galactoside beta-1,3-N-acetylglucosaminyltransferase that catalyzes the transfer of N-acetylglucosamine (GlcNAc) to lactosylceramide, yielding Lc3Cer, the precursor for lacto- and neolacto-series glycosphingolipids. This enzyme acts at a critical branch point in glycan biosynthesis, and its activity is transcriptionally regulated by SP1 and NF-??B, as well as by cytokines and growth factors. B3GNT5 functions upstream of a cascade involving B3GALT5 and FUT7, which further modify the core structure to generate Lewis antigens and sialyl Lewis X??key ligands for E-selectin and P-selectin. These glycosphingolipids mediate selectin-dependent cell adhesion, rolling, and migration, processes central to leukocyte trafficking, cancer metastasis, and inflammatory responses. Interacting directly with the donor substrate UDP-GlcNAc and the acceptor lactosylceramide, B3GNT5 also interfaces with the broader glycosyltransferase network, including B4GALNT1, ST3GAL3, and FUT4.
In HEK293T cells, knockout of B3GNT5 ablates the synthesis of lacto/neolacto-series glycosphingolipids, providing a clean genetic background to dissect the contribution of this glycan branch to cell-surface receptor interactions and signaling. HEK293T cells normally express these glycans, and their elimination allows researchers to attribute changes in cell adhesion, migration, or protein function specifically to the B3GNT5-dependent pathway. Coupled with the SV40 large T antigen, the knockout line can be used for episomal complementation with wild-type or mutant B3GNT5, enabling structure?Cfunction studies. Furthermore, the lack of lacto/neolacto glycosphingolipids simplifies the interpretation of glycoengineering efforts aimed at modulating recombinant protein glycosylation.
Typical applications include probing the role of lacto/neolacto glycosphingolipids in cancer cell adhesion, migration, and invasive behavior, as well as investigating their involvement in inflammatory and autoimmune conditions. The polyclonal knockout population is well-suited for lectin blotting, flow cytometry with glycan-specific lectins (e.g., Lycopersicon esculentum lectin), thin-layer chromatography (TLC) of glycosphingolipids, and mass spectrometry-based glycomics to validate the loss of Lc3Cer and its downstream products. Cell adhesion and migration assays using selectin-coated surfaces or endothelial monolayers can directly assess functional consequences. Additionally, qPCR and Western blotting confirm knockout efficiency at the transcript and protein levels. For further information or to discuss custom knockout strategies, please contact Ascent Research.
