KC-2480

Ba/F3-FGFR3-TACC3-G380R-Cell-Line

24146

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Background of Ba/F3-FGFR3-TACC3-G380R-Cell-Line

FGFR3 is a member of the fibroblast growth factor receptor family, which plays a role in mitogenesis and differentiation. The FGFR3 gene produces various forms of the FGFR3 protein; the location varies depending on the isoform of the FGFR3 protein. Since the different forms are found within different tissues, the protein is responsible for multiple growth factor interactions. Gain of function mutations in FGFR3 inhibits chondrocyte proliferation and underlies achondroplasia and hypochondroplasia.
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 dependent on the activation of the kinases instead of IL-3 supplement. In contrast, their inhibitors can antagonize the kinase-dependent growth effects.

Specifications

Catalog Number	KC-2480
Cell Line Name	Ba/F3-FGFR3-TACC3-G380R-Cell-Line
Host Cell Line	Ba/F3
Description	Stable Ba/F3 cell line expressing exogenous FGFR3-TACC3 fusion gene bearing G380R mutation.
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% 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	Yes

Cell Line Generation

Ba/F3-FGFR3-TACC3-G380R cell line was generated using a lentiviral vector expressing the human FGFR3-TACC3 sequence bearing G380R mutation.

Characterization

Figure1: Characterization of FGFR3-TACC3-G380R in Ba/F3 stable clones using PCR sequencing.

Figure 2:Ba/F3 cells expressing FGFR3-TACC3 mutant were seeded into 96-well plates, treated with compounds for 72 hours, and then read out with Cell-Titer Glo system.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 + 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. FGFR3gene.Genetics Home Reference.U.S.National Library of Medicine. Retrieved 2018-09-27.
2. Wang Y, Liu Z, LiuZ, ZhaoH, zhou X, Cui Y, Han J(May 2013).Advances in research on and diagnosis and treatment of achondroplasia in China. Intractable & Rare Diseases Research. 2(2):45-50. doi:10.5582/irdr.2013.v2.2.45