KC-1024

Ba/F3 ETV6 NTRK2 G639R Cell Line

21401

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Background of Ba/F3 ETV6 NTRK2 G639R Cell Line

NTRK2, also named Tropomyosin receptor kinase B (TrkB), is the high affinity catalytic receptor for the neurotrophin and play a major role in neuronal differentiation and survival, the overactivation or overexpression of NTRK2 fusion protein, due to chromosomal rearrangement, was originally found in congenital fibrosarcoma and subsequently found in secretory breast cancer and other type cancer, the identification of NTRK2 fusion genes as driver genes has lead to the rapid development of anticancer therapeutics agents, such as LOXO-101, PF-026732240, TSR-011, RXDX-101. 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-1024
Cell Line Name	Ba/F3 ETV6 NTRK2 G639R Cell Line
Host Cell Line	Ba/F3
Description	Stable Ba/F3 clone expressing exogenous ETV6-NTRK2 fusion protein bearing G639R mutation in NTRK2 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% 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-ETV6-NTRK2-G639R cell Line was generated using retrovirus vector expressing human ETV6-NTRK2-G639R sequence.

Characterization

Figure 1: Characterization of ETV6-NTRK2 mutation in the Ba/F3 stable clone using PCR sequencing.

Figure 1: 1. Harvest and seed the Ba/F3 cells expressing ETV6-NTRK2-G639R mutant in 96-well plate (3000 cells/90μL medium). 2. Next day, add 10μL 10× serially diluted compound solution each well and incubate the plates for another 72 hours. 3. Add 100μL Cell Titer-Glo each well, mixed and readout using Envision. 4. Plot the dose-responsive curve and fit the IC₅₀ (the centration of 50% inhibition of DMSO vehicle treated clones) using GraphPad Prism software (Version 5).

Cell Resuscitation

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

Cell Freezing

Prepare the freezing medium (70% RPMI-1640 + 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

Vaishnavi, A, A T Le, and R C Doebele. 2015. “TRKing Down an Old Oncogene in a New Era of TargetedTherapy.” Cancer Discovery 5 (1): 25–34.
Jones, David T W, Barbara Hutter, Natalie Jäger, Andrey Korshunov, Marcel Kool, Hans-Jörg Warnatz, Thomas Zichner, et al. 2013. “Recurrent Somatic Alterations of FGFR1 and NTRK2 in Pilocytic Astrocytoma.” Nature
Publishing Group 45 (8). Nature Publishing Group: 927–32.