The CAD Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population targeting the CAD gene. As a heterogeneous pool, these cells provide a loss-of-function model for de novo pyrimidine biosynthesis without clonal bias. They are ideal for investigating nucleotide metabolism, cell proliferation, and pathway regulation.
The HEK293T cell line is derived from human embryonic kidney cells, stably expressing the SV40 large T antigen. This modification enables high episomal plasmid replication, making it a premier system for recombinant protein expression and viral vector production. Its robust growth and transfection efficiency facilitate gene editing studies and metabolic engineering.
CAD encodes a trifunctional enzyme catalyzing the initial, rate-limiting steps of pyrimidine synthesis: converting glutamine, bicarbonate, and ATP into dihydroorotate through carbamoyl phosphate and aspartate intermediates. Its activity is regulated by MAPK/ERK and mTORC1-S6 kinase phosphorylation and UTP allosteric feedback. MYC transcriptionally upregulates CAD. Downstream products include UMP, UTP, CTP, and dTTP. Interactors PRPP synthetase and DHODH further process pathway metabolites.
In HEK293T cells, CAD knockout imposes pyrimidine auxotrophy, compelling reliance on salvage pathways. This is accentuated by the high nucleotide demand from SV40 large T antigen-mediated replication, making the model valuable for studying replication stress and metabolic checkpoints. It serves as a relevant system for cancer metabolism research, where de novo pyrimidine synthesis is often upregulated.
These cells are suited for Western blotting and RT-qPCR to confirm CAD disruption, phospho-CAD analysis, and LC-MS nucleotide pool measurement. Functional assays include proliferation tests under nucleotide limitation and rescue with uridine. The model also enables synergy studies with chemotherapeutics targeting nucleotide synthesis, such as 5-fluorouracil or DHODH inhibitors. For custom inquiries, contact Ascent Research.
