The ALG12 Knockout HT29 Polyclonal Cells constitute a CRISPR/Cas9-edited polyclonal knockout cell population derived from the HT29 human colorectal adenocarcinoma cell line, featuring targeted disruption of the ALG12 gene. This loss-of-function model is designed to enable detailed investigation of ALG12-dependent N-glycosylation processes and associated cellular effects. The polyclonal configuration preserves a heterogeneous knockout background, avoiding the biases of clonal selection while still eliminating ALG12 function across the population.
The parental HT29 cell line is a well-characterized human colorectal adenocarcinoma epithelial model extensively employed in studies of intestinal epithelial biology, including mucin secretion, barrier function, and oncogenic signaling pathways. Derived from a primary colorectal tumor, HT29 cells retain key features of transformed intestinal epithelium, making them particularly suitable for examining glycosylation-dependent processes in the context of colorectal cancer. Their robust growth characteristics and established repertoire of surface glycans provide a relevant platform for assessing changes in glycoprotein expression and cellular behavior.
ALG12 encodes an alpha-1,6-mannosyltransferase that adds the eighth mannose residue to the dolichol-linked oligosaccharide precursor in the endoplasmic reticulum (ER), a critical step in the dolichol cycle of N-glycan biosynthesis. The enzyme functions downstream of earlier ALG mannosyltransferases (ALG1, ALG2, ALG11, ALG3, ALG9) and requires dolichyl-phosphate mannose, provided by the DPM synthase complex (DPM1, DPM2, DPM3), as a sugar donor. ALG12 interacts with ALG9 and other ER-resident mannosyltransferases to complete the lipid-linked oligosaccharide before transfer to nascent proteins by the oligosaccharyltransferase (OST) complex. Its disruption truncates the dolichol intermediate, causing global N-glycoprotein hypoglycosylation and potent induction of the unfolded protein response through sensors IRE1, PERK, and ATF6, thereby reprogramming ER homeostasis and glycoprotein secretion.
In HT29 colorectal cancer cells, ALG12 knockout dissects N-glycosylation roles in malignant intestinal epithelium. Hypoglycosylation of mucins and surface receptors disrupts barrier function and signaling pathways driving tumor progression, invasion, and immune evasion. This model links defective ALG12-dependent glycosylation to ER stress-mediated apoptosis or adaptation, recapitulating aspects of ALG12-congenital disorder of glycosylation type Ig in a cancer context. It also facilitates study of ER quality control and oncogenic signaling intersections, revealing potential therapeutic vulnerabilities.
Key applications include modeling congenital disorders of glycosylation, studying N-glycan truncation effects on colorectal cancer, and screening glycosylation or ER stress modulators. Representative assays include lectin blotting with ConA or L-PHA, flow cytometry for surface glycans using glycosylation-specific antibodies, PNGase F treatment coupled with mass spectrometry glycomics, and immunoblotting for ER stress markers such as BiP and CHOP. Migration/invasion and drug sensitivity assays extend utility in cancer biology research. For technical consultations or custom configurations, please contact Ascent Research.
