KC-1171-DW

Ba/F3-KIF5B-RET-V804M Cell Line

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Background of Ba/F3-KIF5B-RET-V804M Cell Line

RET, abbreviated for "rearranged during transfection" is a receptor tyrosine kinase for membranes of the gial cell line-derived neurotropic neurotrophic factor (GDNF) family of extracellular signaling molecules. Overactivation of RET have associated with a number of cancers. The identification of RET as a driver gene has led to the development of anticancer therapeutics agents.

Specifications

Catalog Number	KC-1171-DW
Cell Line Name	Ba/F3-KIF5B-RET-V804M Cell Line
Clone Number	NA
Host Cell Line	Ba/F3
Description	Stable Ba/F3 clone expressing exogenous KIF5B-RET fusion protein bearing V804M mutation in RET part.
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% RMPI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640+10%FBS
Selection Marker	NA
Morphology	Mostly single, round (some polymorph) cells in suspension
Subculture	Split saturated culture 1:4-1:10 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 20 hours
Mycoplasma Status	Negative

Cell Line Generation

Ba/F3-KIF5B-RET-V804M Cell Line was generated using a retrovirus vector expressing the KIF5B-RET-V804M sequence.

Characterization

Figure 1: Characterization of KIF5B-RET overexpression in the Ba/F3-KIF5B-RET-V804M stable clone using Western Blot.

Figure 2: Characterization of the proliferative effects of Ba/F3-KIF5B-RET-V804M cells and Ba/F3 cells under different concentrations of BLU-667 treatment using the CTG assay.

Figure 3: Validation of in vivo tumorigenicity of Ba/F3 cells stably expressing KIF5B-RET-V804M via subcutaneous implantation in NOD SCID mice, with tumor growth monitored by measuring volume (V=0.5×length×width²) and body weight.

Cell Resuscitation

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

Cell Freezing

Prepare the freezing medium (70% RPMI1640 + 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

Kawamoto Y, Takeda K, Okuno Y, et al. (2004). "Identification of RET autophosphorylation sites by mass spectrometry". J. Biol. Chem. 279 (14): 14213–24.
Rudin, Charles M, Alexander Drilon, and J T Poirier. 2014. “RET Mutations in Neuroendocrine Tumors: Including Small-Cell Lung Cancer.” Journal of Thoracic Oncology 9 (9). Elsevier: 1240–42.
Kohno, Takashi, Koji Tsuta, Katsuya Tsuchihara, Takashi Nakaoku, Kiyotaka Yoh, and Koichi Goto. 2013. “RET Fusion Gene: Translation to Personalized Lung Cancer Therapy.” Cancer Science 104 (11): 1396–1400.