DPH6 Knockout HeLa Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population featuring targeted disruption of the DPH6 gene in the human HeLa cell line. This gene-edited pool provides a loss-of-function model for interrogating DPH6-dependent diphthamide biosynthesis on eukaryotic elongation factor 2 (EEF2). The polyclonal format ensures broad coverage of editing events while avoiding clonal selection bias, facilitating robust functional genomics studies.
The HeLa host cell line originates from a human cervical adenocarcinoma and is positive for human papillomavirus type 18 (HPV18). As an immortalized epithelial cell line, HeLa cells are widely employed in biomedical research, particularly for studying cancer biology, translation regulation, and host?Ctoxin interactions. Their well-characterized genome, rapid proliferation, and ease of manipulation make them an ideal platform for generating CRISPR-based knockouts to investigate post-translational modifications.
DPH6, also known as diphthine??ammonia ligase, functions downstream of DPH5 in the diphthamide biosynthesis pathway. It catalyzes the ATP-dependent amidation of diphthine to diphthamide on a conserved histidine residue of EEF2. This unique modification is essential for EEF2 function and is the target of ADP-ribosylating toxins, including diphtheria toxin and Pseudomonas exotoxin A. The enzymatic reaction requires ATP and ammonia as cofactors, and DPH6 interacts with EEF2, DPH5, and the substrate diphthine. Constitutively expressed, DPH6 acts as the final catalyst in a multi-step pathway involving DPH1?CDPH7, S-adenosyl methionine, and ATP.
Disruption of DPH6 in HeLa cells results in the absence of diphthamide on EEF2, thereby abrogating the ADP-ribosylation that mediates toxin-induced cell death. This model thus provides a powerful tool for dissecting the mechanism of action of bacterial toxins and for investigating the role of diphthamide in translation elongation. Furthermore, the knockout mimics aspects of diphthamide deficiency disorders, which are linked to neurodevelopmental abnormalities, offering a cellular system to study genotype?Cphenotype relationships.
Typical research applications include diphthamide pathway analysis, translation elongation studies, and screening for modulators of toxin sensitivity. Researchers can validate knockout by Sanger sequencing or RT-qPCR, confirm loss of diphthamide modification via western blot or mass spectrometry, and assess functional consequences using diphtheria toxin sensitivity assays or ribosome profiling. Co-immunoprecipitation experiments can probe DPH6?CEEF2 interactions. These polyclonal knockout cells are thus an essential resource for elucidating the biology of this rare post-translational modification and its implications for human disease. For additional details or inquiries, please contact Ascent Research.
