The CCDC22 Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from HT29 human colon adenocarcinoma cells, in which the CCDC22 gene has been disrupted to abolish its expression. This polyclonal pool contains a heterogeneous mixture of loss-of-function alleles, providing a robust model for studying CCDC22-dependent biology without clonal artifacts. The CRISPR/Cas9-mediated gene disruption targets the CCDC22 locus, enabling functional dissection of its role in endosomal trafficking and copper homeostasis.
The parental HT29 cell line is a well-characterized human colon adenocarcinoma epithelial line that retains features of intestinal epithelium, including polarization and enterocytic differentiation markers. Widely employed in studies of intestinal epithelial biology, transport physiology, and oncology, HT29 cells offer a physiologically relevant epithelial context for investigating endosomal recycling and metal transport mechanisms. Disrupting CCDC22 in these cells allows examination of its function in a mucosal barrier model relevant to copper absorption and intestinal homeostasis.
CCDC22 encodes a crucial subunit of the CCC (CCDC93?CCCDC22?CCOMMD) complex, which cooperates with the retromer (VPS35/VPS26/VPS29) and WASH complexes to mediate endosomal sorting and retrograde transport. CCDC22 directly interacts with CCDC93 and COMMD proteins (COMMD1?C10), and this complex is essential for trafficking of ATP7A and LRP1. It facilitates ATP7A translocation from endosomes to the plasma membrane, enabling cellular copper efflux. Loss of CCDC22 disrupts retromer-dependent recycling, impairing ATP7A surface delivery and causing intracellular copper accumulation, which dysregulates copper-dependent enzymes and signaling pathways such as NF-??B. Thus, CCDC22 operates downstream of endocytic uptake and upstream of ATP7A-mediated copper export.
In the HT29 intestinal epithelial model, CCDC22 knockout enables study of impaired copper homeostasis in a mucosal cell type central to dietary copper absorption. This model recapitulates cellular defects observed in copper metabolism disorders and X-linked intellectual disability-epilepsy syndrome linked to CCDC22 mutations. It further allows investigation of interplay between endosomal sorting, copper retention, and inflammatory NF-??B signaling. HT29 cells provide a platform to examine how copper dysregulation affects epithelial integrity, proliferation, and stress responses, connecting intestinal function to systemic copper disorders.
Applications include ATP7A immunofluorescence and flow cytometry for surface ATP7A, copper content quantification by atomic absorption or probes, Western blotting for retromer (VPS35, VPS26) and CCC complex members (CCDC93, COMMD1), endocytosis/recycling assays using transferrin or copper-sensitive dyes, cell viability assays under copper stress, and RT-qPCR for metal-responsive genes. Co-immunoprecipitation can probe disrupted CCC?Cretromer interactions. This tool supports copper homeostasis studies, neurodevelopmental disease modeling, endosomal trafficking investigations, drug screening for metal disorders, and intestinal epithelial biology. For further technical information, contact Ascent Research.
