KC-1552

293T-cyno-CLEC12A-Cell-Line

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Background of 293T-cyno-CLEC12A-Cell-Line

CLEC12A (C-Type Lectin Domain Family 12 Member A) also as so as MICL, it is a Protein Coding gene. CLEC12A/MICL is a cell surface receptor known to regulate signaling cascades and facilitate the tyrosine phosphorylation of target MAP kinases. CLEC12A plays an important immunomodulatory role in AML, CLEC12A is an ideal chimeric antigen receptor T-cell (CAR-T) therapy target for AML and its expression level was closely linked to treatment response and patients' survival outcome. it was highly expressed in AML cell lines and in tissues from AML patients and a higher CLEC12A expression in leukemia stem cells. CLEC12A low expression was associated with poor prognosis in the chemotherapy-only group and high CLEC12A expression may benefit from autologous or allogeneic hematopoietic stem cell transplantation (HSCT). Diseases associated with CLEC12A include Myeloid Leukemia and 3Mc Syndrome.

Specifications

Catalog Number	KC-1552
Cell Line Name	293T-cyno-CLEC12A-Cell-Line
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous cyno CLEC12A gene
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% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 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
In Vivo Validation	NA

Cell Line Generation

293T-cyno-CLEC12A cell line was generated using a lentiviral vector expressing the cyno CLEC12A sequence.

Characterization

Figure 1: Characterization of endogenous cyno CLEC12A expression in 293T cells using QPCR.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 1μg/mL puromycin)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 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 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

Malamud, M., Whitehead, L., McIntosh, A. et al. Recognition and control of neutrophil extracellular trap formation by MICL. Nature 633, 442–450 (2024).
Li Q, Liang C, Xu X, Zhang C, Cao W, Wang M, Jiang Z, Xing H, Yu J. CLEC12A plays an important role in immunomodulatory function and prognostic significance of patients with acute myeloid leukemia. Leuk Lymphoma. 2022 Sep;63(9):2136-2148. doi: 10.1080/10428194.2022.2064986. Epub 2022 Apr 23. PMID: 35481814.
Bill M, Aggerholm A, Kjeldsen E, Roug AS, Hokland P, Nederby L. Revisiting CLEC12A as leukaemic stem cell marker in AML: highlighting the necessity of precision diagnostics in patients eligible for targeted therapy. Br J Haematol. 2019 Mar;184(5):769-781. doi: 10.1111/bjh.15711. Epub 2018 Dec 5. PMID: 30520015.