ITPA Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from the HT29 colorectal adenocarcinoma cell line with targeted disruption of the ITPA gene. This heterogeneous population results from Cas9-mediated gene disruption, providing a versatile loss-of-function model to investigate ITPA-dependent pathways without selection for monoclonal clones.
The HT29 cell line originates from a human primary colorectal adenocarcinoma and is widely employed as an intestinal epithelial cell model. HT29 cells retain key features of the intestinal epithelium, including the ability to differentiate and form polarized monolayers, making them suitable for studies on barrier function, drug absorption, and colorectal cancer biology.
ITPA encodes inosine triphosphate pyrophosphatase, an enzyme that hydrolyzes the non-canonical nucleoside triphosphates ITP and dITP to their corresponding monophosphates, thereby preventing their incorporation into nucleic acids. ITPA functions as a homodimer and is a critical component of the purine metabolism pathway, acting alongside enzymes such as IMPDH, ADA, PNP, and NUDT16 to sanitize the nucleotide pool. By eliminating aberrant nucleotides, ITPA safeguards the fidelity of DNA replication and transcription, and its activity directly influences cellular sensitivity to thiopurine drugs.
In HT29 colorectal adenocarcinoma cells, disruption of ITPA leads to intracellular accumulation of ITP and dITP, which can cause nucleotide pool imbalances, replicative stress, and increased DNA damage??often evidenced by elevated ??H2AX foci. This knockout model recapitulates phenotypic hallmarks of ITPA deficiency, including hypersensitivity to thiopurine-based chemotherapeutics such as 6-mercaptopurine. It therefore provides a physiologically relevant platform to explore the molecular consequences of impaired nucleotide sanitization in an epithelial tumor context.
Researchers can utilize these polyclonal knockout cells to probe mechanisms of nucleotide pool maintenance, DNA replication fidelity, and cellular responses to purine analogs. Typical experimental approaches include Western blotting and RT-qPCR for ITPA expression verification, HPLC or mass spectrometry to quantify ITP/dITP levels, comet assays and ??H2AX immunostaining for DNA damage, and proliferation assays to assess drug sensitivity. Transcriptomic analysis via RNA-seq further reveals downstream gene expression changes. For further information or to discuss this model, contact Ascent Research.
