KC-3995

Ba/F3 EGFR-V769-D770insGGTASV Cell Line

40448

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Background of Ba/F3 EGFR-V769-D770insGGTASV Cell Line

EGFR, the Epidermal growth factor receptor, is a cell-surface receptor tyrosine kinase, and activated by binding of its specific ligand, such as epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha), EGFR overexpression or overactivity have associated with a number of cancers, including the lung cancer and colon cancer. The identification of EGFR as a driver gene has led to the development of anticancer therapeutics agents, including gefitinib, Erlotinib, Afatinib, Osimertinib (AZD9291) and cetuximab. Ba/F3 cell, a murine interleukin-3 dependent pro-B cell line, is a popular system for exploring both kinases and their inhibitors, because some protein kinases can render the Ba/F3 cells to be depended on the activation of the kinases instead of IL-3 supplement, while their inhibitors can antagonize the kinase-dependent growth effects.

Specifications

Catalog Number	KC-3995
Cell Line Name	Ba/F3 EGFR-V769-D770insGGTASV Cell Line
Host Cell Line	Ba/F3
Description	Stable Ba/F3 clone expressing exogenous EGFR gene bearing V769-D770insGGTASV mutation
Quantity	One vial 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% RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10%FBS
Selection Marker	Puromycin
Morphology	Mostly single, round (some polymorph) cells in suspension
Subculture	Split saturated culture 1:10 every 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 20 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

Ba/F3 EGFR-V769-D770insGGTASV cell Line was generated using retrovirus vector expressing EGFR-V769-D770insGGTASV sequence.

Characterization

Figure 1: Characterization of EGFR mutation in the Ba/F3 stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS)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 1:10 every 3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI-1640 + 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. Kobayashi, S. et al. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med 352, 786ÿ792 (2005).

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.