The ITPA Knockout NCI-H1975 Polyclonal Cells are a CRISPR/Cas9-edited heterogeneous population of NCI-H1975 cells harboring targeted disruptions of the ITPA gene. This polyclonal knockout pool is generated by transient introduction of CRISPR/Cas9 components, yielding a diverse array of loss-of-function mutations across the cell population. The product offers a physiologically relevant model to investigate the consequences of ITPA deficiency without the constraints of clonal selection, ensuring broader representation of genetic heterogeneity and functional outcomes.
The NCI-H1975 cell line is a human epithelial cell line derived from a non-small cell lung adenocarcinoma patient. These cells harbor activating EGFR mutations L858R and T790M, which drive constitutive kinase activity and oncogenic signaling, making them a well-established model for EGFR?mutant lung cancer research. The adherent growth and adenocarcinoma origin render NCI-H1975 particularly suitable for studies of tumor cell biology, drug resistance, and targeted therapy evaluation.
ITPA encodes inosine triphosphate pyrophosphatase, an enzyme that hydrolyzes inosine triphosphate (ITP) and deoxyinosine triphosphate (dITP) to their monophosphate forms, thereby preventing incorporation of noncanonical nucleotides into RNA and DNA. This function is essential for preserving nucleotide pool fidelity and genomic integrity. ITPA activity is regulated transcriptionally by NRF2 and is dependent on substrate availability. The enzyme interacts with nucleoside diphosphate kinase (NME1) within the purine salvage pathway. Disruption of ITPA leads to accumulation of ITP and dITP, causing nucleotide pool imbalances that activate DNA damage response kinases ATM and ATR, ultimately promoting mutagenesis. The pathway includes key components such as ITP, IMP, ADA, PNP, NME1, and HPRT, all of which coordinate nucleotide metabolism and cellular stress responses.
In the NCI-H1975 background, ITPA knockout introduces a metabolic vulnerability that can be exploited to probe the intersection of oncogenic signaling and nucleotide homeostasis. Elevated ITP/dITP levels impose replicative stress and DNA damage, which may synergize with the inherent genomic instability of EGFR?mutant adenocarcinoma. This model enables dissection of how purine metabolism modulates sensitivity to nucleoside analog chemotherapeutics, including thiopurines, and may reveal synthetic lethal interactions that are therapeutically tractable.
This polyclonal knockout cell product is ideally suited for a wide range of experimental applications. Researchers can employ Western blotting and RT?qPCR to verify ITPA loss; perform nucleotide pool profiling by HPLC to quantify ITP/dITP accumulation; assess DNA damage via ??H2AX immunofluorescence or comet assay; and evaluate cell viability, apoptosis, and drug sensitivity using MTT/CTG assays and annexin V staining in response to thiopurines or other nucleoside analogs. These applications facilitate investigations into nucleotide metabolism, thiopurine?induced myelotoxicity mechanisms, DNA repair pathway modulation, and preclinical drug testing. For further information or custom inquiries, please contact Ascent Research.
