KC-3791

MDA-MB-231-CRBN-KO Cell Line

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Background of MDA-MB-231-CRBN-KO Cell Line

CRBN encodes a protein related to the Lon protease protein family. It binds to membranes enriched in phosphatidylinositol 4,5-bisphosphate, and shares a conserved modular region termed the epsin NH2-terminal homology (ENTH) domain which plays a crucial role in clathrin-mediated endocytosis through an unknown target. It modifies membrane curvature and facilitates the formation of clathrin-coated invaginations. When epsin function is disrupted, clathrin-mediated endocytosis is blocked.

Specifications

Catalog Number	KC-3791
Cell Line Name	MDA-MB-231-CRBN-KO Cell Line
Clone Number	1B1
Host Cell Line	MDA-MB-231
Description	Stable MDA-MB-231 clone with human CRBN gene knockout, No.1B1
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	NA
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	NA

Cell Line Generation

MDA-MB-231-CRBN-KO-1B1 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of MDA-MB-231-CRBN-KO-1B1 cell line stable clone using PCR sequencing.

Figure 2: Characterization of MDA-MB-231-CRBN-KO-1B1 cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of MDA-MB-231-CRBN-KO-1B1 cell line stable clone using western blot.

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% RPMI-1640 + 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

Wang C, Zhang Y, Wu Y, Xing D. Developments of CRBN-based PROTACs as potential therapeutic agents. Eur J Med Chem. 2021 Dec 5;225:113749. doi: 10.1016/j.ejmech.2021.113749. Epub 2021 Aug 10. PMID: 34411892.
Barankiewicz J, Salomon-Perzyński A, Misiewicz-Krzemińska I, Lech-Marańda E. CRL4CRBN E3 Ligase Complex as a Therapeutic Target in Multiple Myeloma. Cancers (Basel). 2022 Sep 16;14(18):4492. doi: 10.3390/cancers14184492. PMID: 36139651; PMCID: PMC9496858.