The GPR161 Knockout HEK293T Polyclonal Cells product provides a CRISPR/Cas9-edited polyclonal knockout cell population with targeted disruption of the GPR161 gene in the HEK293T human embryonic kidney background. This heterogeneous population of cells harbors diverse loss-of-function mutations, eliminating the need for single-cell cloning and enabling robust functional studies. It is an ideal tool for examining GPR161-dependent signaling processes, particularly those centered on primary cilium-mediated regulation.
HEK293T cells, derived from HEK293 cells by stable integration of SV40 large T antigen, are a standard mammalian host for transient transfection, protein expression, and viral production due to their high transfectability and episomal replication of SV40 origin-containing plasmids. Importantly, these cells can form primary cilia under serum starvation conditions, making them a suitable model system for investigating ciliary signaling dynamics, including the Hedgehog pathway and GPCR trafficking.
GPR161 is an orphan GPCR that constitutively localizes to primary cilia and acts as a negative regulator of Sonic Hedgehog (Shh) signaling. In the cilium, GPR161 couples to G??s and activates adenylyl cyclases, primarily ADCY3, elevating cAMP and stimulating PKA activity. PKA phosphorylates Gli2 and Gli3 transcription factors, promoting their proteolytic processing into repressor forms that inhibit Shh target gene expression. Shh pathway activation triggers removal of GPR161 from cilia via IFT and TULP3-dependent trafficking, reducing cAMP and allowing Gli activator formation. Key downstream mediators include cAMP, PKA, Epac, and Gli2/3, while interacting partners encompass TULP3, IFT complex components, G??s, and ??-arrestin. Thus, GPR161 disruption leads to constitutive Hh pathway activity.
In the HEK293T context, GPR161 knockout provides a simplified, cilia-competent cellular model to dissect the molecular mechanisms of Hedgehog pathway repression and derepression. The loss of GPR161 allows direct assessment of cAMP accumulation, PKA activation, and Gli repressor formation in a system that faithfully recapitulates ciliary signaling without the complexity of multiciliated tissues. This model is particularly valuable for studying ciliopathy-related defects and for high-throughput screening of Hedgehog modulators.
This knockout cell population supports a variety of downstream assays, including cAMP ELISA or FRET-based measurement, Gli-dependent luciferase reporter assays, immunofluorescence microscopy for ciliary protein localization, western blotting for Gli3 repressor isoforms, and RT-qPCR of Shh target genes such as GLI1 and PTCH1. Co-immunoprecipitation can elucidate protein interactions with GPR161 signaling partners. Applications extend to cancer research on SHH-subtype medulloblastoma and basal cell carcinoma, ciliopathy investigation, and drug discovery targeting the Hedgehog pathway. For further information, please contact Ascent Research.
