The DPH5 Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal population derived from HEK293T human embryonic kidney cells. The DPH5 gene has been disrupted to eliminate functional methyltransferase expression, providing a loss-of-function model for diphthamide biosynthesis and translation elongation studies. The polyclonal nature ensures robust target-gene disruption across the culture, minimizing clone-specific artifacts. This format is suitable for assays where mixed genetic backgrounds are acceptable and offers a practical alternative to clonal lines.
HEK293T cells are HEK293 derivatives stably expressing SV40 large T antigen, which promotes episomal plasmid replication and high transfection efficiency. They retain epithelial morphology and are widely used for protein production, viral packaging, and genetic manipulation. The well-characterized HEK293T host facilitates studies of DPH5 function and diphthamide modification in a human cell context.
DPH5 encodes a methyltransferase catalyzing the final step of diphthamide biosynthesis on eEF2??trimethylation of the diphthine intermediate at histidine 715. This conserved modification is critical for eEF2 function in translation elongation. DPH5 functions sequentially with DPH1-4, DPH6, and DPH7 within the pathway. The mature diphthamide is the target of ADP-ribosylation by diphtheria toxin and Pseudomonas exotoxin A, which inactivates eEF2. Loss of DPH5 ablates diphthamide, conferring toxin resistance and enabling functional pathway dissection.
In HEK293T cells, DPH5 knockout facilitates exploration of diphthamide??s role beyond toxin response. Although the precise physiological function is not fully defined, diphthamide is implicated in translational fidelity and ribosomal function under stress. The model is also relevant to DPH5-related neurodevelopmental disorder, characterized by intellectual disability, seizures, and craniofacial dysmorphism. Combined with differentiation protocols, these cells can help elucidate molecular mechanisms underlying this condition.
These polyclonal knockout cells support diverse assays including western blotting for eEF2 and diphthamide modification, mass spectrometry, RT-qPCR for DPH5 transcript, and diphtheria toxin sensitivity testing. Growth assays can reveal metabolic or translational defects, and ADP-ribosylation assays provide mechanistic detail. The mixed genetic background also aids drug screening for diphthamide biosynthesis modulators or toxin inhibitors. For customized inquiries, contact Ascent Research.
