KC-4195

293T-FGFR3IIIC-S249C Cell Line

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Background of 293T-FGFR3IIIC-S249C Cell Line

FGFR3 IIIc (Fibroblast Growth Factor Receptor 3 isoform IIIc), also known as FGFR3c, CD333, or JTK4, is a receptor tyrosine kinase predominantly expressed in mesenchymal cells. The S249C mutation results from a serine-to-cysteine substitution at position 249, causing ligand-independent dimerization and constitutive activation of the receptor. This hotspot mutation is most prevalent in non‑muscle‑invasive bladder cancer, detected in up to 74% of low‑grade pTa tumors, and is also associated with thanatophoric dysplasia type I. The pan‑FGFR inhibitor erdafitinib is FDA‑approved for metastatic urothelial carcinoma harboring S249C, while novel FGFR3‑selective inhibitors like dabogratinib (TYRA-300) are in clinical development.

Specifications

Catalog Number	KC-4195
Cell Line Name	293T-FGFR3IIIC-S249C Cell Line
NCBI/UniProt Accession Number	NM_000142
Clone Number	2-1#
Host Cell Line	293T Cell Line
Description	Stable 293T cell line expressing exogenous human FGFR3IIIc gene with the S249C mutation
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
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

The 293T-FGFR3IIIc-S249C cell line was generated using a lentiviral vector expressing the FGFR3IIIc sequence harboring the S249C mutation.

Characterization

Figure 1: Characterization of human FGFR3IIIC-S249C overexpression in the 293T-FGFR3IIIC-S249C stable clone using FACS.

Figure 2: Characterization of human FGFR3IIIC-S249C overexpression in the 293T-FGFR3IIIC-S249C stable clone using sequencing.

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 following day.

References

1.Kelleher, Fergal C et al. “Fibroblast growth factor receptors, developmental corruption and malignant disease.” Carcinogenesis vol. 34,10 (2013): 2198-205. doi:10.1093/carcin/bgt254.
2. Li, Daosheng et al. “Ipsilateral synchronous papillary renal neoplasm with reverse polarity and urothelial carcinoma in a renal transplant recipient: a rare case report with molecular analysis and literature review.” Diagnostic pathology vol. 18,1 120. 3 Nov. 2023, doi:10.1186/s13000-023-01405-w.

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.