EML4-ALK, echinoderm microtubule-associated protein-like 4 - anaplastic lymphoma kinase, is an abnormal protein with transforming activity found in some primary lung malignant tumors due to the fusion of abnormal configuration of DNA wherein the echinoderm microtubule-associated protein-like 4 (EML4) gene is fused to the anaplastic lymphoma kinase (ALK) gene; EML4-ALK and its mutants can promote and maintain the malignant behavior of the cancer cells. Identifying ALK as a driver gene has led to the rapid development of anticancer therapeutic agents, including crizotinib, ceritinib, and Brigatinib.
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.
Cell Line Name:
Ba/F3 EML4-ALK-G1202R-L1196M Cell Line
Host Cell Line:
Mouse Ba/F3 cell line
Stable Ba/F3 clone expressing exogenous EML4-ALK fusion gene with G1202R-L1196M point mutation in the ALK part.
One vial of frozen cells (5X106 per vial)
Stable in culture over a minimum of 10 passages
Drug screening and biological assays
70% RPMI1640+20% FBS+10% DMSO
Mostly single, round (some polymorph) cells in suspension
Split saturated culture 1:10 every 3 days; seed out at about 1-3 × 105 cells/mL
37 °C with 5% CO2
Liquid nitrogen immediately upon receiving
Approximately 20 hours
Cell Line Generation
Ba/F3 EML4-ALK-G1202R-L1196M cell Line was generated using retrovirus vector expressing human EML4-ALK G1202R-L1196M sequence.
Characterization using PCR sequencing and WB
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.
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
1. Koivunen, J. P. et al. EML4-ALK Fusion Gene and Efficacy of an ALK Kinase Inhibitor in Lung Cancer. Clinical Cancer Research 14, 4275–4283 (2008).
2. Choi, Y. L. et al. Identification of Novel Isoforms of the EML4-ALK Transforming Gene in Non-Small Cell Lung Cancer. Cancer Research 68, 4971–4976 (2008).
3. Gainor, J. F. et al. Molecular Mechanisms of Resistance to First- and Second-Generation ALK Inhibitors in ALK Rearranged Lung Cancer. Cancer Discovery 1–48 (2016). doi:10.1158/2159-8290.CD-16-059
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