The ATP7B Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal cell population in which the ATP7B gene has been disrupted, generating a loss-of-function model for studying copper homeostasis and Wilson disease in an intestinal epithelial framework. This heterogeneous pool of gene-edited HT29 cells avoids the limitations of clonal selection and provides a robust system for investigating ATP7B-dependent copper transport and downstream molecular consequences.
The host HT29 cell line originates from a human colorectal adenocarcinoma and displays adherent epithelial morphology. It is a well-established model for intestinal epithelial barrier function, expressed ample transporter proteins, and forms polarized monolayers with tight junctions. These attributes render HT29 particularly suited for examining the role of ATP7B in copper handling within the gastrointestinal tract, where copper absorption and excretion are tightly regulated.
ATP7B encodes a copper-transporting P-type ATPase that pumps copper from the cytosol into the trans-Golgi network for incorporation into cuproenzymes such as ceruloplasmin and also facilitates biliary copper excretion. Transcription of ATP7B is driven by SP1 and the metal-responsive factor MTF1 in response to cellular copper levels. The protein interacts with the copper chaperone ATOX1, which delivers copper to ATP7B, and with COMMD1, a regulator of copper export. Downstream, ATP7B activity loads copper onto ceruloplasmin and promotes metallothionein expression, while the copper importer SLC31A1 (CTR1) balances cellular uptake. Disruption of ATP7B thus uncouples the copper trafficking network.
Knockout of ATP7B in HT29 cells alters copper homeostasis, likely impairing cuproenzyme biogenesis and rendering cells more susceptible to copper-induced toxicity. This model is instrumental for dissecting intestinal copper absorption mechanisms and the molecular pathology of Wilson disease, as it mirrors the copper accumulation and biliary excretion defects observed in hepatocytes. Moreover, the HT29 background allows assessment of how colon adenocarcinoma cells cope with copper overload or chelation, providing insights into metal-related cancer cell vulnerabilities.
Research applications include modeling Wilson disease in a intestinal epithelial context, investigating copper transport kinetics, screening copper chelators or ionophores, and evaluating copper-mediated cytotoxicity. Typical assays encompass Western blot and RT-qPCR for gene expression validation, ICP-MS for cellular copper quantification, 64Cu uptake and efflux measurements, MTT viability assays, immunofluorescence for ATP7B localization, and RNA-seq for transcriptome-wide copper-response profiling. For further details, please contact Ascent Research.
