The C1GALT1 Knockout HEK293T Cell Line is a CRISPR/Cas9-mediated gene-disrupted derivative of the HEK293T human embryonic kidney epithelial cell line, designed for loss-of-function studies of core 1 synthase glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase 1. This knockout cell line enables interrogation of mucin-type O-glycan biosynthesis and the consequences of ablating core 1 O-glycan formation (T antigen). By eliminating C1GALT1, it allows dissection of O-glycosylation-dependent processes in a host background widely used for recombinant protein expression and viral production.
HEK293T cells originate from HEK293 cells, immortalized from primary human embryonic kidney cells with sheared adenovirus 5 DNA, and additionally express SV40 large T antigen for episomal replication of SV40 origin-containing plasmids. This adherent epithelial line is noted for high transfection efficiency, robust protein production, and utility in lentiviral/retroviral packaging, making it an optimal host for gene disruption and subsequent overexpression or rescue experiments.
C1GALT1 encodes T-synthase, which transfers galactose from UDP-galactose to N-acetylgalactosamine-O-Ser/Thr residues to form core 1 O-glycan (T antigen), a key step in extending mucin-type O-glycans beyond the Tn antigen. Its activity requires the specific chaperone COSMC and interaction with HSP90B1 (GRP94) in the endoplasmic reticulum, while SP1 transcriptionally regulates C1GALT1 expression. Knockout of C1GALT1 causes accumulation of Tn antigen on substrates such as mucins MUC1 and MUC16, integrins, receptor tyrosine kinases, and E-cadherin, disrupting glycoprotein stability, cell adhesion, and downstream signaling.
In the HEK293T context, C1GALT1 knockout provides a defined system to study how mucin-type O-glycosylation modulates glycoprotein trafficking, cell?Ccell/matrix interactions, and immune recognition. This model is particularly relevant in cancer biology, where aberrant O-glycosylation with Tn antigen expression drives tumor progression via altered adhesion and immune evasion, and also aids in investigating Tn syndrome and IgA nephropathy. HEK293T??s manipulability supports complementation with C1GALT1 mutants and COSMC co-expression to validate chaperone dependence.
Typical applications include lectin blotting with PNA and flow cytometry for Tn/T antigens, glycoproteomics by mass spectrometry, and functional assays such as cell adhesion, migration, immunoprecipitation, western blotting of glycosylated proteins, and RT-qPCR to confirm C1GALT1 disruption. The cell line is essential for dissecting glycosylation-dependent signaling in oncogenic contexts and for evaluating therapeutics targeting aberrant glycosylation. For further information or to discuss custom applications, please contact Ascent Research.
