KC-4103

EMT6-mCD47-KO-2A2-Cell-Line

41815

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Background of EMT6-mCD47-KO-2A2-Cell-Line

The CD47 gene in mice (officially named Cd47, located on chromosome 2 in Mus musculus) encodes a heavily glycosylated cell surface protein that interacts with its ligand, signal regulatory protein alpha (SIRPα), to regulate critical cellular processes including immune cell function, apoptosis, and cell proliferation. Specifically, Cd47 overexpression on cancer cells effectively inhibits phagocytosis by macrophages, thereby promoting tumor growth and metastasis. Investigations using genetically modified mouse models have elucidated the therapeutic potential of targeting Cd47 in cancer therapy. For example, blocking Cd47-SIRPα interactions using monoclonal antibodies led to enhanced macrophage-mediated tumor cell phagocytosis and tumor regression in preclinical models of various cancers. Further research has explored the complex interplay between Cd47 and other immune checkpoint regulators, revealing potential synergistic effects when combined in cancer immunotherapy regimensThese insights underscore the multifaceted nature of Cd47 biology and its attractiveness as a therapeutic target.

Specifications

Catalog Number	KC-4103
Cell Line Name	EMT6-mCD47-KO-2A2-Cell-Line
Host Cell Line	EMT6
Description	Stable EMT6 clone with Mouse CD47 gene knockout, No.2A2
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	Epithelial
Subculture	Split saturated culture 1:4-1:5 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	Yes

Cell Line Generation

EMT6-mCD47-KO 2A2 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of EMT6-mCD47-KO 2A2 Cell Line stable clone using FACS.

Figure 2: Characterization of EMT6-mCD47-KO 2A2 cell line stable clone using PCR sequencing.

Figure 3: Characterization of EMT6-mCD47-KO 2A2 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 1:4-1:5 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

Willingham SB, Volkmer JP, Gentles AJ, Sahoo D, Dalerba P, Mitra SS, Wang J, Contreras-Trujillo H, Martin R, Cohen JD, Lovelace P, Scheeren FA, Chao MP, Weiskopf K, Tang C, Volkmer AK, Naik TJ, Storm TA, Mosley AR, Edris B, Schmid SM, Sun CK, Chua MS, Murillo O, Rajendran P, Cha AC, Chin RK, Kim D, Adorno M, Raveh T, Tseng D, Jaiswal S, Enger PØ, Steinberg GK, Li G, So SK, Majeti R, Harsh GR, van de Rijn M, Teng NN, Sunwoo JB, Alizadeh AA, Clarke MF, Weissman IL. The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors. Proc Natl Acad Sci U S A. 2012 Apr 24;109(17):6662-7. doi: 10.1073/pnas.1121623109. Epub 2012 Mar 26. PMID: 22451913; PMCID: PMC3340046.