KC-3880

293T-mouse-GFRAL Cell Line

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Background of 293T-mouse-GFRAL Cell Line

Growth Differentiation Factor 15 (GDF-15), also called Macrophage inhibitory cytokine-1 (MIC-1), is a divergent member of the transforming growth factor beta (TGF-beta) superfamily. GFRAL (GDNF receptor-alpha-like) is a functional receptor of GDF15, facilitating weight-loss functions of the protein through c-Ret downstream signaling. GDF-15 and GFRAL signaling pathway is implicated in diet-based obesity and insulin resistance.

Specifications

Catalog Number	KC-3880
Cell Line Name	293T-mouse-GFRAL Cell Line
Clone Number	22
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous mouse-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-mouse-GFRAL cell line was generated using lentivirus expressing mouse-GFRAL sequence.

Characterization

Figure 1. Characterization of mouse-GFRAL over-expression in the 293T-mouse-GFRAL stable clone using qPCR.

Figure 2. Characterization of mouse-GFRAL over-expression in the 293T-mouse-GFRAL stable clone using PCR.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS, 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

Bootcov MR, Bauskin AR, Valenzuela SM, Moore AG, Bansal M, He XY, et al.(October 1997). MIC-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF-beta superfamily. Proceedings of the National Academy of Sciences of the United States of America. 94 (21): 11514–9.
Zimmers TA, Jin X, Hsiao EC, McGrath SA, Esquela AF, Koniaris LG (June 2005).Growth differentiation factor-15/macrophage inhibitory cytokine-1 induction after kidney and lung injury. Shock. 23 (6): 543–8.