KC-3933

293T-FGFR2-KO Cell Line

40334

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Background of 293T-FGFR2-KO Cell Line

The fibroblast growth factor receptor (FGFR) family is a family of tyrosine kinase receptors that include FGFR1, FGFR2, FGFR3, and FGFR4. Following the binding of growth factors, FGFRs dimerize and activate intracellular signaling pathways responsible for cellular proliferation, survival, and angiogenesis . The FGFR2 gene contains at least 24 exonic sequences, however, only subgroups of these are used for different isoforms through alternative splicing. To the present day, more than 25 isoforms of FGFR2 have been described .The binding of an FGF and heparin/heparan sulfates as co-factors to FGFR2 results in dimerization and subsequently trans autophosphorylation of the receptor at its cytoplasmic component. The activated intracellular kinase domain phosphorylates downstream targets, leading to the activation of numerous signaling pathways, including JAK-STAT, MAPK, and PI3K-AKT.

Specifications

Catalog Number	KC-3933
Cell Line Name	293T-FGFR2-KO Cell Line
Clone Number	2A3
Host Cell Line	293T
Description	Stable 293T cell line with human FGFR2 gene knockout
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
Selection Marker	NA
Morphology	Fibroblastoid cells growing as a monolayer
Subculture	Split the saturated culture at a ratio of 1:4~1:5 every 2~3 days; seed out at about 1-3 x 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-FGFR2-KO cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of 293T-FGFR2-KO Cell Line stable clone using PCR sequencing.

Figure 2: Characterization of293T-FGFR2-KO Cell Line stable clone using RT-PCR sequencing.

Figure 3: Characterization of 293T-FGFR2-KO Cell Line stable clone using Western blot.

Cell Resuscitation

Prewarm culture medium (DMEM+10% FBS)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 1:4-1:5 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

Angerilli V, Fornaro L, Pepe F, Rossi SM, Perrone G, Malapelle U, Fassan M. FGFR2 testing in cholangiocarcinoma: translating molecular studies into clinical practice. Pathologica. 2023 Apr;115(2):71-82. doi: 10.32074/1591-951X-859. Epub 2023 Apr 5. PMID: 37017301; PMCID: PMC10462997.