KC-5529

293T-CRE-Luc2-cyno-GCGR Cell Line

66255

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Background of 293T-CRE-Luc2-cyno-GCGR Cell Line

Glucagon receptor (GCGR, alias GGR) is a class B GPCR mainly expressed in the liver. Its physiological function is to stimulate hepatic gluconeogenesis and glycogenolysis via the Gαs-cAMP-CREB signaling pathway to maintain blood glucose balance. Excessive GCGR activation leads to hyperglucagonemia, closely linked to type 2 diabetes, obesity and fatty liver disease. Multiple GCGR-targeted agents are under development, including small-molecule antagonists, anti-GCGR antibodies and GLP-1R/GCGR dual agonists that have advanced to late-stage clinical trials. Distinct interspecies differences exist between human, rodent and cynomolgus GCGR, so primate-derived GCGR assays are essential for translational pharmacology. The 293T-CRE-Luc2-cyno-GCGR stable cell line stably expresses cynomolgus monkey GCGR and CRE-controlled Luc2 reporter. Ligand-induced GCGR activation elevates intracellular cAMP to drive luciferase expression, producing measurable luminescence. This model supports high-throughput functional assessment of GCGR agonists, antagonists and inverse agonists with non-human primate pharmacological relevance.

Specifications

Catalog Number	KC-5529
Cell Line Name	293T-CRE-Luc2-cyno-GCGR Cell Line
NCBI/UniProt Accession Number	XM_045376476.1, XM_005585255.2
Clone Number	5#
Host Cell Line	293T-CRE-Luc2
Description	Stable 293T-CRE-Luc2 cell line stably overexpressing cynomolgus GCGR gene.
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% basal medium+20% FBS+10% DMSO
Propagation Medium	DMEM+10%FBS+1µg/mL puromycin
Selection Marker	puromycin
Morphology	Epithelial-like
Subculture	Split saturated culture 1:4-1:8 every 2-3 days
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

293T-CRE-Luc2-cyno-GCGR cell line was generated using a lentiviral vector expressing cynomolgus GCGR sequence.

Characterization

Cell Resuscitation

Pre-warm complete culture medium (basal medium and 10% FBS) in a 37°C water bath.
Rapidly thaw the cryovial in a 37°C water bath for 1-2 minutes with gentle agitation.
Transfer the vial to a biosafety cabinet, and disinfect the exterior with 70% ethanol.
Aseptically transfer the cell suspension dropwise into a sterile centrifuge tube containing 9.0 mL of pre-warmed complete medium.
Centrifuge at approximately 125 × g for 5–7 minutes at room temperature, carefully aspirate the supernatant without disturbing the cell pellet.
Gently resuspend the pellet in an appropriate volume of complete medium and transfer the suspension into a T25 flask.
Incubate the flask in a 37°C in a humidified 5% CO₂ incubator.
Assess cell viability and morphology after 24 hours. If cells appear healthy, replace the medium with fresh medium supplemented with the appropriate selective antibiotic.
Subculture the cells at a ratio of 1:4-1:8 every 2-3 days upon reaching 80%–90% confluency.

Cell Freezing

Prepare the freezing medium (70% basal medium, 20% FBS and 10% DMSO) freshly before use.
Pre-chill the freezing medium on ice and label the cryovials accordingly.
Transfer the cell suspension to a sterile conical tube and perform a cell count to determine total viability and density.
Centrifuge the cells at 250×g for 5 minutes at room temperature; carefully aspirate the supernatant.
Gently resuspend the cell pellet in chilled freezing medium, ensuring a minimum cell density of 3×10⁶ cells/mL.
Aliquot 1 mL of the cell suspension into each pre-labeled cryovial.
Place the cryovials into a CoolCell® container and store at -80°C overnight for controlled-rate cooling.
Transfer the cryovials to the liquid nitrogen for long-term storage the folHighing day.

References

Zhou C, Dhall D, Nissen NN, Chen CR, Yu R. Homozygous P86S mutation of the human glucagon receptor is associated with hyperglucagonemia, alpha cell hyperplasia, and islet cell tumor. Pancreas. 2009 Nov;38(8):941-6. doi: 10.1097/MPA.0b013e3181b2bb03. PMID: 19657311; PMCID: PMC2767399.
Hilton JF, Christensen KE, Watkins D, Raby BA, Renaud Y, de la Luna S, Estivill X, MacKenzie RE, Hudson TJ, Rosenblatt DS. The molecular basis of glutamate formiminotransferase deficiency. Hum Mutat. 2003 Jul;22(1):67-73. doi: 10.1002/humu.10236. Erratum in: Hum Mutat. 2003 Nov;22(5):416. PMID: 12815595.
Horn F, Weare J, Beukers MW, Hörsch S, Bairoch A, Chen W, Edvardsen O, Campagne F, Vriend G. GPCRDB: an information system for G protein-coupled receptors. Nucleic Acids Res. 1998 Jan 1;26(1):275-9. doi: 10.1093/nar/26.1.275. PMID: 9399852; PMCID: PMC147194.

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.