KC-4743

CHOK1-FGFR2b-EGFP-Middle Cell Line

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Home » CHOK1-FGFR2b-EGFP-Middle Cell Line

Background of CHOK1-FGFR2b-EGFP-Middle Cell Line

FGFR2 (Fibroblast Growth Factor Receptor 2) is a Protein Coding gene. 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.Green fluorescent protein is a protein composed of about 238 amino acids, which can be excited from blue light to ultraviolet light, and emits green fluorescence. Green fluorescent protein (GFP), commonly referred to as enhanced green fluorescent protein (EGFP), is a widely used variant of wild-type green fluorescent protein (wt-GFP). EGFP, or Enhanced Green Fluorescent Protein, and GFP, which stands for Green Fluorescent Protein. EGFP is a GFP mutant line, and the most widely used is the mutant of GFP: enhanced green fluorescent protein (EGFP) (64-bit phenylpropylene bright), which emits fluorescence intensity more than 6 times greater than that of GFP.

Specifications

Catalog NumberKC-4743
Cell Line NameCHOK1-FGFR2b-EGFP-Middle Cell Line
Host Cell LineCHOK1
DescriptionCHOK1 cell line stably expressing exogenous FGFR2b-EGFP gene
QuantityTwo vials of frozen cells (≥2-106/vial)
StabilityStable in culture over a minimum of 10 passages
ApplicationDrug screening and biological assays
Freezing Medium70% RPMI1640 + 20% FBS + 10% DMSO
Propagation MediumRPMI1640 + 10% FBS + 10 μg/mL Puromycin
Selection MarkerPuromycin
MorphologyFibroblastoid cells growing as monolayer
SubcultureSplit saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-2 × 105 cells/mL
Incubation37 °C with 5% CO2
StorageLiquid nitrogen immediately upon receiving
Doubling TimeApproximately 24 hours
Mycoplasma StatusNegative

Cell Line Generation

CHOK1-FGFR2b-EGFP-Middle cell line was generated using a lentiviral vector expressing the FGFR2b EGFP sequence.

Characterization

Figure 1: Figure 1: Characterization of FGFR2b EGFP overexpressing in CHOK1 stable clones using FACS.

Figure2: Characterization of CHOK1-FGFR2b-EGFP-Middle cell line stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 10 μg/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 9.0 mL 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 1:4-1:8 every 2-3 days; seed out at about 1-2 × 105 cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI1640 + 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. Byron SA, Chen H, Wortmann A, Loch D, Gartside MG, Dehkhoda F, Blais SP, Neubert TA, Mohammadi M, Pollock PM. The N550K/H mutations in FGFR2 confer differential resistance to PD173074, dovitinib, and ponatinib ATP-competitive inhibitors. Neoplasia. 2013 Aug;15(8):975-88. doi: 10.1593/neo.121106. PMID: 23908597; PMCID: PMC3730048.
2. Morris LM, Klanke CA, Lang SA, Lim FY, Crombleholme TM. TdTomato and EGFP identification in histological sections: insight and alternatives. Biotech Histochem. 2010 Dec;85(6):379-87. doi: 10.3109/10520290903504753. Epub 2010 Jan 29. PMID: 20109099.
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