KC-1170-DW

Ba/F3-KIF5B-RET-V804L Cell Line

58358

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Background of Ba/F3-KIF5B-RET-V804L 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-1170-DW
Cell Line Name	Ba/F3-KIF5B-RET-V804L Cell Line
Clone Number	NA
Host Cell Line	Ba/F3
Description	Stable Ba/F3 clone expressing exogenous KIF5B-RET fusion protein bearing V804L mutation in RET part.
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% 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-V804L Cell Line was generated using a retrovirus vector expressing the KIF5B-RET-V804L sequence.

Characterization

Figure 1: Characterization of KIF5B-RET overexpression in the Ba/F3-KIF5B-RET-V804L stable clone using Western Blot.

Figure 2: Characterization of the proliferative effects of Ba/F3-KIF5B-RET-V804L 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-V804L 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.

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.