KC-4430

Ba/F3-BCR-ABL-M244V-Cell-Line

46463

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Background of Ba/F3-BCR-ABL-M244V-Cell-Line

The ABL1 gene is an oncogene located on the long arm of chromosome 9 at band q34 (9q34). Its gene product is a non-receptor tyrosine protein kinase. The BCR gene is located on the long arm of chromosome 22 at band q11 (22q11). Due to a translocation between the long arms of chromosomes 9 and 22, the ABL oncogene on chromosome 9 (9q34) and the BCR (B-cell receptor) gene on chromosome 22 (22q11) are recombined to form the BCR-ABL fusion gene. The BCR/ABL fusion gene is an anti-apoptotic gene with high tyrosine kinase activity, which causes excessive cell proliferation and leads to dysregulation of cellular control. 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-4430
Cell Line Name	Ba/F3-BCR-ABL-M244V-Cell-Line
Host Cell Line	Ba/F3
Description	Stable Ba/F3 clone expressing exogenous BCR-ABL gene bearing M244V 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	NA

Cell Line Generation

Ba/F3 BCR-ABL-M244V cell Line was generated using retrovirus vector expressing human BCR-ABL-M244V sequence.

Characterization

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

Figure 2: Characterization of IC50 (Half Inhibitory Concentration) in Ba/F3 BCR-ABL-M244V stable clone using GraphPad Prism software.

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 1:10 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

Shibata N, Ohoka N, Hattori T, Naito M. Development of a Potent Protein Degrader against Oncogenic BCR-ABL Protein. Chem Pharm Bull (Tokyo). 2019;67(3):165-172. doi: 10.1248/cpb.c18-00703. PMID: 30827996.
Burke BA, Carroll M. BCR-ABL: a multi-faceted promoter of DNA mutation in chronic myelogeneous leukemia. Leukemia. 2010 Jun;24(6):1105-12. doi: 10.1038/leu.2010.67. Epub 2010 May 6. PMID: 20445577; PMCID: PMC4425294.