KC-3782

Ba/F3-MPL-CALR-del52

Stable Ba/F3 cell line expressing exogenous MPL & CALR-del52 gene

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Background of Ba/F3-MPL-CALR-del52

MPL (Thrombopoietin Receptor), also known as CD110 or TPOR, is a cytokine receptor primarily expressed on hematopoietic stem cells and megakaryocytes. CALR (Calreticulin) is an ER chaperone protein. The CALR del52 (type 1 mutation) is a 52-bp deletion in exon 9, generating a frameshift and a novel C-terminal amino acid sequence. This mutation is a driver in 67% of JAK2/MPL‑unmutated essential thrombocythemia (ET) and 88% of primary myelofibrosis (PMF). Mechanistically, mutant CALR binds directly to MPL, causing ligand‑independent JAK‑STAT activation. Current therapies include JAK inhibitors (e.g., ruxolitinib); emerging agents directly targeting mutant CALR, such as INCA033989, are in clinical development.

Specifications

Catalog Number	KC-3782
Cell Line Name	Ba/F3-MPL-CALR-del52
NCBI/UniProt Accession Number	NM_005373, NM_004343.4
Clone Number	1#
Host Cell Line	Mouse Ba/F3 cell line
Description	Stable Ba/F3 cell line expressing exogenous human MPL and del52 mutant form of CALR 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
Selection Marker	Puromycin \| G418
Morphology	Mostly single, round (some polymorph) cells in suspension
Subculture	Split saturated culture 1:10 every 3 days
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 20 hours
Mycoplasma Status	Negative
In Vivo Validation	Yes

Cell Line Generation

Ba/F3-MPL-CALR-del52 cell line was generated using a retrovirus expressing the human MPL and del52 mutant form of CALR sequence.

Characterization

Figure: Characterization of BaF3-MPL-CALR-del52 Cell Line stable clone using PCR sequencing.

Cell Resuscitation

Pre-warm complete culture medium (basal medium and 10% FBS) in a 37°C water bath.
Rapidly thaw the cryovial in a 37°C water bath for 1-2 minutes with gentle agitation.
Transfer the vial to a biosafety cabinet, and disinfect the exterior with 70% ethanol.
Aseptically transfer the cell suspension dropwise into a sterile centrifuge tube containing 9.0 mL of pre-warmed complete medium.
Centrifuge at approximately 125 × g for 5–7 minutes at room temperature, carefully aspirate the supernatant without disturbing the cell pellet.
Gently resuspend the pellet in an appropriate volume of complete medium and transfer the suspension into a T25 flask.
Incubate the flask in a 37°C in a humidified 5% CO₂ incubator.
Assess cell viability and morphology after 24 hours. If cells appear healthy, replace the medium with fresh medium supplemented with the appropriate selective antibiotic.
Subculture the cells at a ratio of 1:10 every 3 days upon reaching 80%–90% confluency.

Cell Freezing

Prepare the freezing medium (70% basal medium, 20% FBS and 10% DMSO) freshly before use.
Pre-chill the freezing medium on ice and label the cryovials accordingly.
Transfer the cell suspension to a sterile conical tube and perform a cell count to determine total viability and density.
Centrifuge the cells at 250×g for 5 minutes at room temperature; carefully aspirate the supernatant.
Gently resuspend the cell pellet in chilled freezing medium, ensuring a minimum cell density of 3×10⁶ cells/mL.
Aliquot 1 mL of the cell suspension into each pre-labeled cryovial.
Place the cryovials into a CoolCell® container and store at -80°C overnight for controlled-rate cooling.
Transfer the cryovials to the liquid nitrogen for long-term storage the following day.

References

1. Lim, K-H et al. “Expression of CALR mutants causes mpl-dependent thrombocytosis in zebrafish.” Blood cancer journal vol. 6,10 e481. 7 Oct. 2016, doi:10.1038/bcj.2016.83.
2. Toppaldoddi, Katte Rao et al. “Rare type 1-like and type 2-like calreticulin mutants induce similar myeloproliferative neoplasms as prevalent type 1 and 2 mutants in mice.” Oncogene vol. 38,10 (2019): 1651-1660. doi:10.1038/s41388-018-0538-z.
3. Nieborowska-Skorska, Margaret et al. “Ruxolitinib-induced defects in DNA repair cause sensitivity to PARP inhibitors in myeloproliferative neoplasms.” Blood vol. 130,26 (2017): 2848-2859. doi:10.1182/blood-2017-05-784942.

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.