The CCDC91 Knockout HT29 Polyclonal Cells comprise a CRISPR/Cas9-edited polyclonal population of human HT29 colorectal adenocarcinoma cells with targeted disruption of the CCDC91 gene. This knockout model provides a versatile loss-of-function tool for dissecting the gene’s function in intracellular protein sorting and membrane trafficking. The polyclonal format ensures a diverse pool of edited cells, enabling robust population-level analyses without the biases of clonal selection, and is ideal for studying Golgi-to-endosome transport and lysosomal enzyme delivery in cancer contexts.
The HT29 cell line, derived from a primary colorectal adenocarcinoma of a 44-year-old Caucasian female, is a well-established in vitro model for intestinal epithelial biology and colorectal cancer. These adherent epithelial cells retain oncogenic mutations and key signaling pathways, offering a physiologically relevant host for investigating membrane trafficking alterations associated with malignancy.
CCDC91 encodes a coiled-coil domain-containing adaptor protein that functions at the trans-Golgi network (TGN) downstream of the small GTPase ARF1. It directly interacts with Golgi-localized, ??-ear-containing, ARF-binding (GGA) adaptor proteins GGA1 and GGA2, thereby recruiting clathrin and the adaptor protein complex AP-1. This molecular assembly packages mannose 6-phosphate receptors (MPRs) into transport vesicles destined for endosomes, a step crucial for the proper delivery of lysosomal enzymes and the recycling of MPRs. Additional pathway components include Rab GTPases and early endosomes. Disruption of CCDC91 therefore impairs the TGN-to-endosome trafficking axis, compromising lysosomal biogenesis and cellular homeostasis.
In the HT29 colorectal cancer background, loss of CCDC91 is predicted to disturb endolysosomal function by blocking the normal sorting of lysosomal hydrolases, leading to potential defects in autophagy, nutrient sensing, and drug sequestration. These processes are intimately linked to cancer cell survival, proliferation, and resistance to chemotherapeutic agents. Consequently, this polyclonal knockout model serves as a powerful system to interrogate the contribution of CCDC91-mediated trafficking to colorectal cancer pathophysiology and to explore the interplay between intracellular transport and oncogenic signaling.
The CCDC91 Knockout HT29 Polyclonal Cells can be employed in a broad range of experimental applications, including Western blotting and RT-qPCR for knockout confirmation and expression analysis of downstream effectors such as MPRs and lysosomal enzymes. Immunofluorescence microscopy facilitates visualization of Golgi and endosome marker redistribution. Functional investigations encompass lysosomal enzyme activity assays, clathrin-mediated endocytosis assays, and flow cytometry for surface receptor quantification. Cell viability and drug sensitivity assays can reveal the impact on therapeutic responses. For additional technical details, please contact Ascent Research.
