KC-4865

Ba/F3-EPOR-JAK2 Cell Line

53354

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Background of Ba/F3-EPOR-JAK2 Cell Line

EPOR encodes the erythropoietin receptor which is a member of the cytokine receptor family. Upon erythropoietin binding, this receptor activates Jak2 tyrosine kinase which activates different intracellular pathways including: Ras/MAP kinase, phosphatidylinositol 3-kinase and STAT transcription factors. The stimulated erythropoietin receptor appears to have a role in erythroid cell survival. Defects in the erythropoietin receptor may produce erythroleukemia and familial erythrocytosis. Dysregulation of this gene may affect the growth of certain tumors. Alternate splicing results in multiple transcript variants.
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-4865
Cell Line Name	Ba/F3-EPOR-JAK2 Cell Line
Clone Number	2#
Host Cell Line	Ba/F3
Description	Stable Ba/F3 clone expressing exogenous EPOR-JAK2 gene
Quantity	One vial 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+8ng/mL EPO
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

Cell Line Generation

Ba/F3 EPOR-JAK2 cell Line was generated using retrovirus vector expressing EPOR-JAK2 sequence.

Characterization

Figure 1: Characterization of EPOR-JAK2 in the Ba/F3 stable clone using PCR sequencing.

Figure 2: Characterization of EPOR-JAK2 in the Ba/F3 stable clone using Western blot.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 8ng/mL EPO)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. Takeda J, Yoshida K, Nakagawa MM, Nannya Y, Yoda A, Saiki R, Ochi Y, Zhao L, Okuda R, Qi X, Mori T, Kon A, Chiba K, Tanaka H, Shiraishi Y, Kuo MC, Kerr CM, Nagata Y, Morishita D, Hiramoto N, Hangaishi A, Nakazawa H, Ishiyama K, Miyano S, Chiba S, Miyazaki Y, Kitano T, Usuki K, Sezaki N, Tsurumi H, Miyawaki S, Maciejewski JP, Ishikawa T, Ohyashiki K, Ganser A, Heuser M, Thol F, Shih LY, Takaori-Kondo A, Makishima H, Ogawa S. Amplified EPOR/JAK2 Genes Define a Unique Subtype of Acute Erythroid Leukemia. Blood Cancer Discov. 2022 Sep 6;3(5):410-427. doi: 10.1158/2643-3230.BCD-21-0192. PMID: 35839275; PMCID: PMC9894574.

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.