KC-2421

293T-cyno-GFRAL-Cell-Line

24026

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Background of 293T-cyno-GFRAL-Cell-Line

GDNF-family receptor α-like, GFRAL (also known as GRAL) belongs to the Glial Cell Line Derived Neurotrophic Factor (GDNF) family of α-like receptors.GFRAL is a type I transmembrane protein, and activation of GFRAL can regulate metabolism, reduce body weight, and improve blood glucose, and will be useful in the treatment of severe obesity, type 2 diabetes, and anorexia/malignant disease. Recent studies have shown that GFRAL-mediated signaling mechanisms are associated with cancer, and GFRAL may be useful as a biomarker for the initial stages of underlying pathologies in the diagnosis, treatment, and efficacy evaluation of many diseases.

Specifications

Catalog Number	KC-2421
Cell Line Name	293T-cyno-GFRAL-Cell-Line
Host Cell Line	293T
Description	Stable HEK293T clone expressing exogenous cyno GFRAL 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% 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
In Vivo Validation	NA

Cell Line Generation

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

Characterization

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

Cell Resuscitation

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

Cell Freezing

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

References

Breit SN, Brown DA, Tsai VW. The GDF15-GFRAL Pathway in Health and Metabolic Disease: Friend or Foe? Annu Rev Physiol. 2021 Feb 10;83:127-151. doi:10.1146/annurev-physiol-022020-045449. Epub 2020 Nov 23. PMID: 33228454.
Dimeric arrangement of the parathyroid hormone receptor and a structural mechanism for ligand-induced dissociation. PMID: 20172855 PMCID: PMC2852981 DOI: 10.1074/jbc.M109.093138.
Suriben R, Chen M, Higbee J, Oeffinger J, Ventura R, Li B, Mondal K, Gao Z, Ayupova D, Taskar P, Li D, Starck SR, Chen HH, McEntee M, Katewa SD, Phung V, Wang M, Kekatpure A, Lakshminarasimhan D, White A, Olland A, Haldankar R, Solloway MJ, Hsu JY, Wang Y, Tang J, Lindhout DA, Allan BB. Antibody-mediated inhibition of GDF15-GFRAL activity reverses cancer cachexia in mice. Nat Med. 2020 Aug;26(8):1264-1270. doi: 10.1038/s41591-020-0945-x. Epub 2020 Jul 13. PMID: 32661391.