KC-3098

NIH3T3-FGFR2-BICC1-K714R Cell Line Cell Line

26621

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Background of NIH3T3-FGFR2-BICC1-K714R Cell Line Cell Line

The protein encoded by this gene is a member of the fibroblast growth factor receptor family, where amino acid sequence is highly conserved between members and throughout evolution. FGFR family members differ from one another in their ligand affinities and tissue distribution. A full-length representative protein consists of an extracellular region, composed of three immunoglobulin-like domains, a single hydrophobic membrane-spanning segment and a cytoplasmic tyrosine kinase domain. The extracellular portion of the protein interacts with fibroblast growth factors, setting in motion a cascade of downstream signals, ultimately influencing mitogenesis and differentiation. This particular family member is a high-affinity receptor for acidic, basic and/or keratinocyte growth factor, depending on the isoform. Mutations in this gene are associated with Crouzon syndrome, Pfeiffer syndrome, Craniosynostosis, Apert syndrome, Jackson-Weiss syndrome, Beare-Stevenson cutis gyrata syndrome, Saethre-Chotzen syndrome, and syndromic craniosynostosis. Multiple alternatively spliced transcript variants encoding different isoforms have been noted for this gene.

Specifications

Catalog Number	KC-3098
Cell Line Name	NIH3T3-FGFR2-BICC1-K714R Cell Line Cell Line
Host Cell Line	NIH/3T3
Description	Stable NIH/3T3 clone expressing exogenous FGFR2 gene bearing BICC1-K714R mutation.
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 + 2μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Fibroblast
Subculture	Split saturated culture 1:4-1:6 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

NIH3T3-FGFR2-BICC1-K714R cell line was generated using a lentiviral vector expressing the human FGFR2-BICC1-K714R sequence.

Characterization

Figure 1: Characterization of human FGFR2-BICC1-K714R in the NIH/3T3 stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS and 2μg/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 1:4-1:6 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. Vogel A, Segatto O, Stenzinger A, Saborowski A. FGFR2 Inhibition in Cholangiocarcinoma. Annu Rev Med. 2023 Jan 27;74:293-306.
2. Goyal L, Meric-Bernstam F, Hollebecque A, Valle JW, Morizane C, Karasic TB, Abrams TA, Furuse J, Kelley RK, Cassier PA, Klümpen HJ, Chang HM, Chen LT, Tabernero J, Oh DY, Mahipal A, Moehler M, Mitchell EP, Komatsu Y, Masuda K, Ahn D, Epstein RS, Halim AB, Fu Y, Salimi T, Wacheck V, He Y, Liu M, Benhadji KA, Bridgewater JA; FOENIX-CCA2 Study Investigators. Futibatinib for FGFR2-Rearranged Intrahepatic Cholangiocarcinoma. N Engl J Med. 2023 Jan 19;388(3):228-239.