KC-5128

Ba/F3-Mouse-JAK2-V617F-KI Cell Line

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Background of Ba/F3-Mouse-JAK2-V617F-KI Cell Line

Janus kinase 2 (JAK2) is a critical non-receptor tyrosine kinase involved in cytokine signaling through the JAK-STAT pathway. In mice, JAK2 mediates responses to hematopoietic cytokines such as erythropoietin (EPO), thrombopoietin (TPO), and interleukins, playing essential roles in hematopoiesis, immune regulation, and cell proliferation. The constitutive activation of JAK2, particularly via the V617F mutation, is associated with myeloproliferative neoplasms (MPNs), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. Mouse models with JAK2 mutations have been instrumental in studying MPN pathogenesis and testing targeted therapies, such as JAK2 inhibitors (e.g., ruxolitinib). Additionally, JAK2 knockout studies in mice demonstrate its necessity for embryonic development, particularly in definitive erythropoiesis. Despite advances in understanding JAK2’s role, challenges remain in developing therapies that selectively inhibit mutant JAK2 without disrupting normal cytokine signaling. Further research using murine models is crucial for elucidating JAK2’s broader functions and improving therapeutic strategies for JAK2-related disorders.

Specifications

Catalog Number	KC-5128
Cell Line Name	Ba/F3-Mouse-JAK2-V617F-KI Cell Line
Clone Number	1A3
Host Cell Line	Ba/F3
Description	Stable Ba/F3 clone expressing endogenous Mouse JAK2 gene bearing V617F mutations, No.1A3
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	RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10% FBS
Selection Marker	N/A
Morphology	Mostly single, round (some polymorph) cells in suspension
Subculture	Split saturated culture 1:4-1:6 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 30 hours
Mycoplasma Status	Negative

Cell Line Generation

Ba/F3-Mouse-JAK2-V617F-KI cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of Ba/F3-Mouse-JAK2-V617F-KI cell line stable clone using PCR sequencing.

Figure 2: Characterization of Ba/F3-Mouse-JAK2-V617F-KI cell line stable clone using RT-PCR sequencing.

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 1:4-1:6 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

Parganas, E., et al. (1998). Jak2 is essential for signaling through a variety of cytokine receptors. Cell, 93(3), 385-395.
Baxter, E.J., et al. (2005). Acquired mutation of JAK2 in myeloproliferative disorders. The Lancet, 365(9464), 1054-1061.
Mullally, A., et al. (2010). Physiological Jak2V617F expression causes a lethal myeloproliferative neoplasm with differential effects on hematopoietic stem and progenitor cells. Cancer Cell, 17(6), 584-596.