The GNAS Knockout HEK293T Polyclonal Cells constitute a CRISPR/Cas9-edited polyclonal knockout cell population originating from the HEK293T human embryonic kidney cell line. This heterogeneous pool carries diverse indel mutations in the GNAS gene, which encodes the Gs?? subunit of stimulatory heterotrimeric G proteins, leading to loss of functional protein. The product is generated by transient delivery of CRISPR/Cas9 components targeting critical exons, yielding a mixed population suitable for bulk functional studies without clonal selection. It represents a representative loss-of-function model for GNAS, preserving population-level diversity.
HEK293T cells are a derivative of the 293 line, stably expressing SV40 large T antigen, which enables episomal plasmid replication and high transfection efficiency. These adherent, epithelial-like cells are commonly used for transient and stable expression, as well as viral production. Their robust heterologous protein expression and well-characterized signaling networks make them ideal for GPCR pharmacology and functional genomics. The human cellular background provides a relevant context for studying disease-related signaling pathways.
The GNAS locus encodes the Gs?? protein, a ubiquitously expressed GTP-binding subunit that couples activated heptahelical receptors to adenylyl cyclase, stimulating cAMP production. Gs?? is activated by numerous GPCRs, including ??-adrenergic, thyroid-stimulating hormone, and parathyroid hormone receptors. Upon activation, Gs?? exchanges GDP for GTP, dissociates from G?¦?, and directly activates adenylyl cyclase isoforms, leading to a rise in intracellular cAMP. Elevated cAMP activates PKA, which phosphorylates targets such as CREB, thereby modulating gene expression. Gs?? signaling also intersects with MAPK/ERK, Wnt/??-catenin, and calcium pathways. The GNAS transcript undergoes alternative splicing and imprinting, producing additional isoforms like XL??s and NESP55, but Gs?? is the predominant form.
In HEK293T cells, GNAS disruption eliminates the primary pathway for GPCR-triggered cAMP accumulation, providing a null background to parse Gs??-dependent signaling from G??i- or G??q-mediated events. The knockout model facilitates characterization of disease-associated GNAS mutations underlying McCune-Albright syndrome, pseudohypoparathyroidism, and fibrous dysplasia. Given the endogenous GPCR expression in HEK293T, these cells serve as a clean platform for reconstitution with wild-type or mutant Gs?? variants, studying orphan receptors, and evaluating cAMP-independent G?¦? signaling.
Applications include receptor pharmacology profiling with cAMP accumulation assays, GPCR agonist dose-response studies, phospho-CREB immunoblotting, and CRE-luciferase reporter assays. The knockout pool is useful for genome-wide CRISPR screens, crosstalk studies between cAMP and other second messengers, and drug discovery targeting Gs??-coupled receptors. Researchers can also perform calcium mobilization assays and co-immunoprecipitation to map altered signaling networks. For additional technical specifications, lot-specific editing data, and culture guidance, please contact Ascent Research.
