The ALG3 Knockout HT29 Polyclonal Cells represent a CRISPR/Cas9-edited polyclonal cell population derived from the HT29 cell line, in which the ALG3 gene has been disrupted. This pool of edited cells provides a heterogeneous loss-of-function model for studying alpha-1,3-mannosyltransferase function in a human colorectal adenocarcinoma background.
HT29 is a well-established human epithelial cell line isolated from a 44-year-old female with Dukes?? stage B colorectal adenocarcinoma. Widely used as a model for intestinal epithelial differentiation and colorectal cancer, these cells retain the capacity to polarize and form tight junctions in vitro, making them suitable for studies of epithelial biology and glycosylation-dependent processes in the gut mucosa.
ALG3 encodes an alpha-1,3-mannosyltransferase localized to the endoplasmic reticulum membrane, where it catalyzes the transfer of the first mannose residue from dolichol phosphate mannose to the Man5GlcNAc2-PP-dolichol intermediate. This enzymatic step is essential for the assembly of the lipid-linked oligosaccharide precursor required for N-glycosylation. ALG3 function is tightly linked to the unfolded protein response (UPR); under ER stress, transcription factors such as XBP1s and ATF4 upregulate ALG3 expression. Downstream, ALG3 activity directly impacts the glycosylation and subsequent folding of numerous client proteins, including epidermal growth factor receptor (EGFR) and integrins, which depend on proper N-glycosylation for membrane localization and signaling. ALG3 works in concert with ALG9 and ALG12 to extend the oligosaccharide chain, and its substrate is provided by the dolichol phosphate mannose synthase complex.
Disruption of ALG3 in the HT29 colorectal adenocarcinoma background creates a powerful tool for dissecting the role of N-glycosylation in cancer-associated processes. Aberrant glycosylation is a hallmark of many malignancies, influencing tumor cell adhesion, migration, and immune evasion. In these knockout polyclonal cells, diminished ALG3 activity is expected to perturb the glycan profile of key oncogenic receptors and adhesion molecules, potentially altering downstream signaling pathways such as EGFR-mediated proliferation. Additionally, the model can be used to simulate aspects of ALG3-congenital disorder of glycosylation (ALG3-CDG) in an epithelial context, enabling investigation of how glycosylation defects intersect with epithelial barrier function and tumorigenesis. The polyclonal population preserves a range of knockout alleles, reflecting genetic heterogeneity relevant to research.
Researchers can employ these ALG3 knockout polyclonal cells for a wide range of glycosylation-focused investigations. Lectin blotting with concanavalin A (ConA) or high-resolution LC-MS-based glycan profiling can characterize N-glycan alterations, while lectin-based flow cytometry allows quantitative assessment of surface glycosylation. N-glycosylation site mapping by mass spectrometry enables identification of specific sites affected by ALG3 loss on proteins such as EGFR or integrins. Activation of ER stress pathways can be monitored by measuring CHOP and BiP expression via RT-qPCR, and tunicamycin sensitivity can be assessed in viability assays. These cells also serve as a platform for screening glycosylation modulators, supporting drug discovery for glycosylation-dependent cancer. For further details, please contact Ascent Research.
