The AMPD2 Knockout HT29 Polyclonal Cells comprise a CRISPR/Cas9-edited polyclonal knockout population of the human colorectal adenocarcinoma cell line HT29, engineered to disrupt the gene encoding adenosine monophosphate deaminase 2 (AMPD2). This product provides a loss-of-function model generated through CRISPR/Cas9-mediated gene disruption, resulting in a heterogeneous pool of cells with targeted inactivation of AMPD2 alleles. As a polyclonal product, it captures diverse editing outcomes across the population, enabling robust and reproducible functional studies without the limitations of single clone selection. The knockout model is suited for investigating purine nucleotide metabolism, energy homeostasis, and oncogenic signaling networks in a colorectal cancer background.
The parental HT29 cell line is a well-characterized human colorectal adenocarcinoma model displaying epithelial morphology and widely employed in cancer biology, drug discovery, and metabolic research. HT29 cells are known for their ability to form polarized monolayers, express intestinal markers, and respond to metabolic perturbations, making them a pertinent host for dissecting purine nucleotide cycle dynamics. Their origin from a primary colon tumor renders them genetically relevant for colorectal cancer investigations, and their well-documented sensitivity to chemotherapeutic agents provides a practical platform for drug sensitivity screens. The epithelial nature of HT29 cells further facilitates assays involving cell adhesion, migration, and metabolic flux analyses.
AMPD2 encodes the homotetrameric enzyme adenosine monophosphate deaminase 2, which catalyzes the deamination of AMP to IMP, a critical step in the purine nucleotide cycle. This reaction is tightly coupled to the regulation of intracellular adenine and guanine nucleotide pools and is influenced by cellular energy status, as reflected by the AMP/ATP ratio. Upstream regulators include the cellular energy sensor AMPK and the transcription factor MYC, which putatively modulates AMPD2 expression. Downstream, IMP production feeds into purine nucleotide synthesis and contributes to ammonia generation. AMPD2 activity directly impacts AMPK signaling, as changes in AMP levels alter AMPK phosphorylation and activity, thereby influencing ATP/GTP homeostasis. Representative pathway components include adenylosuccinate synthetase (ADSS) and adenylosuccinate lyase (ADSL), which act sequentially downstream of AMPD2 in the cycle. The disruption of AMPD2 perturbs these intricate interactions, providing a model to interrogate the AMPK?Cpurine metabolism axis.
In the HT29 colorectal cancer context, AMPD2 knockout abrogates adenosine monophosphate deaminase activity, leading to accumulation of AMP and reduced IMP production, thereby dysregulating the purine nucleotide cycle and altering energy-sensing pathways. This disruption can impair AMPK-mediated metabolic reprogramming, which is often co-opted by cancer cells to support rapid proliferation. The model thus enables the study of how colorectal adenocarcinoma cells adapt to nucleotide imbalance, potentially revealing vulnerabilities in purine metabolism or compensatory mechanisms such as enhanced salvage pathway activity. Additionally, as AMPD2 mutations are linked to neurological disorders like pontocerebellar hypoplasia type 6 and hereditary spastic paraplegia 63, this knockout tool may offer insights into the metabolic underpinnings of these diseases, particularly when combined with neuronal differentiation protocols. However, the primary utility remains rooted in cancer metabolism research, where HT29 cells serve as a tractable system.
Researchers can employ this knockout model to investigate purine metabolism in colorectal cancer, examine AMPK signaling dynamics under metabolic stress, and assess the role of AMPD2 in tumor cell proliferation, survival, and drug sensitivity. The polyclonal population is suitable for quantitative Western blotting to confirm depletion of AMPD2 and assess total AMPK?? and phosphorylated AMPK levels, as well as HPLC-based quantitation of cellular nucleotides (AMP, IMP, ATP, GTP). Metabolic flux analyses using Seahorse technology can measure alterations in oxygen consumption and glycolytic rates, while luminescence-based AMP/ATP ratio assays provide rapid assessment of energy status. Drug sensitivity assays with antimetabolites such as methotrexate or 5-fluorouracil can identify synthetic lethal interactions, and transwell migration assays may explore AMPD2’s impact on metastatic potential. These applications collectively facilitate deep functional annotation of AMPD2 in colorectal cancer. For additional technical information, product availability, or custom inquiries, please contact Ascent Research.
