Lgr5 Knockout ATDC-5 Cell Line

The Lgr5 Knockout ATDC-5 Cell Line is a CRISPR/Cas9-edited knockout cell line originating from the mouse ATDC-5 chondrogenic cell line. This loss-of-function model targets the Lgr5 gene, which encodes the leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), known to serve as a receptor for R-spondins and potentiate Wnt/??-catenin signaling. By disrupting Lgr5, researchers can investigate the role of R-spondin-LGR5 interactions in modulating canonical Wnt pathway activity without interference from endogenous Lgr5 expression. The cell line is supplied as a validated knockout line suitable for a variety of cellular and molecular assays in signaling and differentiation research.

The ATDC-5 cell line was originally derived from mouse teratocarcinoma cells and has been extensively characterized as a chondrogenic progenitor model. It recapitulates the multistep process of chondrogenesis, transitioning from mesenchymal condensation to hypertrophic chondrocytes, and is widely used to study endochondral bone formation. ATDC-5 cells express extracellular matrix components such as type II collagen and aggrecan under differentiation conditions, making them a physiologically relevant host for interrogating pathways that govern cartilage development and skeletal biology.

LGR5 functions as a high-affinity receptor for R-spondin family members (RSPO1, RSPO2, RSPO3, RSPO4). Upon R-spondin binding, LGR5 sequesters the transmembrane E3 ubiquitin ligases RNF43 and ZNRF3, which normally target Frizzled receptors for degradation. This sequestration prevents Frizzled ubiquitination, leading to increased receptor availability at the cell surface and potentiation of Wnt ligand binding. Consequently, Dishevelled (DVL) is activated, ??-catenin stabilizes and accumulates, and nuclear translocation promotes TCF/LEF-mediated transcription of target genes such as AXIN2, MYC, and CCND1. Knockout of Lgr5 abolishes this R-spondin-dependent enhancement, thereby attenuating downstream Wnt/??-catenin signaling and reducing expression of proliferation-associated targets.

In the ATDC-5 chondrogenic context, Lgr5 expression is associated with a progenitor-like state and may influence the balance between self-renewal and differentiation. Wnt/??-catenin signaling has dual roles, promoting early chondrogenesis while inhibiting terminal hypertrophic differentiation. Disrupting Lgr5 provides a means to dissect how R-spondin/LGR5-mediated signal potentiation specifically contributes to these transitions. This knockout cell line thus enables detailed examination of LGR5-dependent mechanisms in chondroprogenitor maintenance, cartilage formation, and the crosstalk between Wnt signaling and chondrogenic regulators.

This knockout model is recommended for a broad range of research applications, including investigation of Wnt/RSPO signaling in chondrogenesis, stem cell maintenance, colorectal cancer (where LGR5 is a well-known cancer stem cell marker), and tissue regeneration studies. Researchers can employ representative assays such as western blot analysis of ??-catenin and AXIN2, TOP/FOP flash reporter assays to measure Wnt pathway activity, RT-qPCR quantification of Wnt target genes, immunofluorescence staining for stem cell markers, flow cytometry for LGR5 expression, and chondrogenesis differentiation assays using insulin-transferrin-selenium-based media. For further details or to request technical support, please contact Ascent Research.