This product consists of a CRISPR/Cas9-edited polyclonal knockout cell population derived from the HT29 colorectal adenocarcinoma cell line, targeting the GPAA1 gene. The polyclonal format provides a heterogeneous pool of cells harboring diverse loss-of-function mutations, enabling robust population-level studies of GPI anchor biosynthesis without clonal selection artifacts. This configuration is well-suited for high-throughput phenotypic screening and assays where genetic heterogeneity mimics tumor variability.
HT29 is a widely used human colorectal adenocarcinoma cell line, originally established from a primary tumor. These epithelial cells are tumorigenic in xenograft models and exhibit differentiation features under specific culture conditions, expressing intestinal markers such as mucins and brush border enzymes. HT29 serves as a key model for colorectal cancer research, intestinal epithelial biology, and drug development, offering a relevant background to investigate pathways dysregulated in tumorigenesis.
GPAA1 encodes an essential subunit of the GPI transamidase complex, which also includes PIG-K, PIG-S, PIG-T, and PIG-U. This ER-resident complex cleaves C-terminal signal peptides from nascent proteins and transfers them to preassembled GPI anchors. GPAA1 is thought to contribute to the catalytic mechanism of this transamidation reaction. Transcription factors SP1 and NF-Y regulate expression of GPI biosynthesis genes, while mTOR and PERK/eIF2??/ATF4 pathways modulate the pathway under nutrient and ER stress conditions. GPI anchoring is required for surface expression of numerous proteins, including CD55, CD59, uPAR, glypicans, and the Wnt co-receptors LGR5 and RSPO receptors. Consequently, GPAA1 knockout results in intracellular accumulation and degradation of these GPI-anchored proteins, disrupting Wnt/??-catenin signaling, cell adhesion, and complement regulation.
In the HT29 background, GPAA1 knockout provides a model to study the role of GPI-anchored proteins in colorectal cancer. Loss of surface LGR5 and RSPO receptors attenuates Wnt/??-catenin signaling, potentially reducing stemness and proliferation. Depletion of CD55 and CD59 sensitizes cells to complement-mediated lysis, offering insights into immune evasion mechanisms. Furthermore, reduced uPAR surface levels impair urokinase-dependent proteolysis and cell migration, enabling investigation of metastatic potential. This model also recapitulates features of GPI biosynthesis disorders, such as paroxysmal nocturnal hemoglobinuria, in an epithelial context, facilitating drug screening for agents that bypass GPI deficiency or exploit vulnerabilities of GPI-AP loss.
Standard applications include flow cytometry to quantify surface CD55 and CD59, western blotting for GPI-APs and GPAA1, and Wnt/??-catenin luciferase reporter assays. Migration and invasion can be assessed using transwell chambers. The polyclonal population can be adapted for xenograft tumor studies and co-immunoprecipitation of the GPI transamidase complex. These cells are also a starting point for generating clonal knockout lines. For inquiries, please contact Ascent Research.
