The GNPTG Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout population derived from the human HT29 colorectal adenocarcinoma cell line. This product provides a heterogeneous knockout model through targeted disruption of GNPTG, avoiding single-cell cloning. Researchers can use these cells to investigate loss-of-function phenotypes related to the gamma subunit of GlcNAc-1-phosphotransferase and the mannose 6-phosphate (M6P) trafficking pathway. The polyclonal format ensures broad representation of gene-editing events, suitable for studies where clonal variation is undesirable.
HT29 cells, originating from a primary colorectal adenocarcinoma of a 44-year-old Caucasian female, serve as a widely adopted model for colon cancer research and intestinal epithelial biology. Their well-documented proliferation and differentiation characteristics provide a robust platform for gene-editing applications. This GNPTG knockout model leverages the HT29 background to explore how defective lysosomal enzyme sorting influences colorectal cancer cell behavior, offering insights into the interplay between lysosomal function and tumor pathology.
GNPTG encodes the gamma subunit of the heterohexameric GlcNAc-1-phosphotransferase complex, which recognizes lysosomal hydrolases and catalyzes the initial step of M6P tag addition. It cooperates with GNPTAB (alpha/beta subunits) and is transcriptionally regulated by TFEB. The M6P tag binds MPR300 and MPR46 receptors, directing enzymes such as Cathepsin D, Cathepsin B, and Hexosaminidase A to lysosomes. Knockout disrupts efficient M6P formation, causing missorting and hypersecretion of a subset of hydrolases. Interacting factors include clathrin adaptors AP-1 and GGAs.
In the HT29 colorectal cancer context, GNPTG knockout facilitates investigation of lysosomal contribution to cancer progression. Lysosomes regulate autophagy, nutrient sensing, and secretion of matrix-remodeling enzymes, all relevant to tumor malignancy. This model enables study of how mistargeted hydrolases affect proliferation and invasion. It also serves as a cellular disease model for mucolipidosis III gamma. Validation can be performed via Cathepsin D immunoblotting, LAMP1 immunofluorescence, and Lysotracker staining to assess lysosomal pH and enzyme activity.
Applications include dissecting lysosomal enzyme sorting, modeling mucolipidosis III gamma, and screening for small-molecule modulators. Researchers can employ fluorometric enzyme activity assays, M6P western blotting, ELISAs for secreted hydrolases, and cell proliferation assays. It is ideal for studying lysosomal dysfunction in colorectal cancer and exploring therapeutic interventions. For additional technical information, please contact Ascent Research.
