The DPM1 Knockout HEK293T Polyclonal Cells represent a CRISPR/Cas9-edited polyclonal knockout cell population derived from the human embryonic kidney HEK293T cell line, designed for functional ablation of the DPM1 gene. This product provides a heterogeneous pool of edited cells with targeted disruption of DPM1, avoiding clonal isolation artifacts and enabling robust analysis of gene function within a polyclonal context. The knockout model serves as a critical tool for investigating glycosylation-dependent pathways without the constraints of single-clone variability, offering a representative landscape of loss-of-function phenotypes.
HEK293T cells are an immortalized human embryonic kidney epithelial cell line constitutively expressing the SV40 large T antigen. This feature facilitates episomal amplification of plasmids containing the SV40 origin of replication, leading to high-level transient protein expression and efficient viral production. The robust translational machinery and well-characterized secretome of HEK293T make it an ideal host for studying post-translational modifications, particularly glycosylation, as well as for applications demanding reproducible expression of complex glycoproteins and viral envelope proteins.
DPM1 encodes the catalytic subunit of the dolichol-phosphate mannose (Dol-P-Man) synthase complex, which resides on the cytoplasmic face of the endoplasmic reticulum and forms a stable heterotrimer with DPM2 and DPM3. This complex transfers mannose from GDP-mannose to dolichol phosphate, generating Dol-P-Man??the obligate mannose donor for N-linked glycosylation, O-mannosylation, and glycosylphosphatidylinositol (GPI) anchor biosynthesis. Upstream, DPM1 expression is regulated by the ER stress sensors ATF6 and XBP1, linking its activity to the unfolded protein response. Downstream, Dol-P-Man supplies mannose residues to ALG glycosyltransferases for oligosaccharide assembly, the OST complex for en bloc transfer to nascent proteins, and GPI biosynthetic enzymes, thereby determining the maturation of diverse glycoproteins and GPI-anchored proteins.
In the HEK293T background, DPM1 knockout disrupts the early steps of glycosylation, leading to impaired N-glycan processing, deficient O-mannosylation, and reduced cell surface expression of GPI-anchored proteins such as CD59. This glycosylation deficiency can trigger ER stress and perturb protein folding, making the model valuable for recapitulating molecular signatures of congenital disorder of glycosylation type Ie (CDG Ie). Furthermore, HEK293T??s capacity for high-level expression of recombinant proteins allows detailed dissection of glycoprotein trafficking defects and the interplay between glycosylation and cell adhesion or signaling pathways.
The polyclonal knockout population is suitable for a wide array of research applications, including investigation of N-linked glycosylation deficiencies using western blotting for glycosylation-sensitive proteins (e.g., ICAM-1, Lamp-1) and lectin blotting with ConA or L-PHA. Flow cytometric analysis of GPI-anchored proteins such as CD59 provides a direct readout of GPI anchor synthesis, while complement-mediated cytotoxicity assays assess functional GPI anchoring. Dolichol-phosphate mannose synthase activity can be quantified via metabolic labeling with [2-3H]mannose, and transcriptomic changes in glycosylation-related genes can be profiled by RNA-seq. Additional applications encompass drug screening for glycosylation modulators, evaluation of viral glycoprotein processing, and mechanistic studies of ER stress-dependent signaling. For further information, please contact Ascent Research.
