KC-5450

293T-GCGR-Cell-Line

61159

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Background of 293T-GCGR-Cell-Line

GCGR encoded by this gene is the glucagon receptor, a member of the secretin receptor family of class B G-protein-coupled receptors (GPCRs). This receptor plays a critical role in maintaining blood glucose homeostasis by mediating the effects of glucagon, including stimulation of hepatic glycogenolysis and gluconeogenesis. This gene is expressed primarily in the liver and kidney but also in the heart, adrenal glands, pancreas, spleen, thymus, cerebral cortex, adipose tissue, and gastrointestinal tract. Mutations in this gene can impair glucagon signaling, leading to dysregulation of glucose metabolism and contributing to the development of non-insulin-dependent diabetes mellitus (type 2 diabetes). Such defects may result in altered glucose production, insufficient counter-regulation during fasting, and increased susceptibility to hyperglycemia. Inactivating mutations in this gene cause resistance to glucagon which is associated with Mahvash disease.

Specifications

Catalog Number	KC-5450
Cell Line Name	293T-GCGR-Cell-Line
NCBI/UniProt Accession Number	NM_000160.4
Clone Number	11#
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous GCGR gene
Quantity	One vial of frozen cells (≥2×10⁶ cells)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative

Cell Line Generation

293T-GCGR-cell-line was generated using a lentiviral vector expressing the GCGR sequence.

Characterization

Figure 1: Characterization of GCGR overexpression in the 293T-GCGR stable clone using FACS.

Figure 2: Characterization of GCGR in the 293T-GCGR stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS and 1μg/mL puromycin)in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO₂ incubator.
8. Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage.

References

1.McGlone ER, Ansell TB, Dunsterville C, Song W, Carling D, Tomas A, Bloom SR, Sansom MSP, Tan T, Jones B. Hepatocyte cholesterol content modulates glucagon receptor signalling. Mol Metab. 2022 Sep;63:101530. doi: 10.1016/j.molmet.2022.101530. Epub 2022 Jun 16. PMID: 35718339; PMCID: PMC9254120.
2.Kotliar IB, Bendes A, Dahl L, Chen Y, Saarinen M, Ceraudo E, Dodig-Crnković T, Uhlén M, Svenningsson P, Schwenk JM, Sakmar TP. Multiplexed mapping of the interactome of GPCRs with receptor activity-modifying proteins. Sci Adv. 2024 Aug 2;10(31):eado9959. doi: 10.1126/sciadv.ado9959. Epub 2024 Jul 31. PMID: 39083597; PMCID: PMC11290489.

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.