KC-5816

Ba/F3-mCRBN-humanized-KI-Plus Cell Line

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Background of Ba/F3-mCRBN-humanized-KI-Plus Cell Line

The Ba/F3-mCRBN-humanized-KI cell line is generated by introducing specific humanizing point mutations into the endogenous mouse Crbn locus. This model is designed to support targeted protein degradation research, particularly the high-throughput screening of molecular glue degraders and the investigation of their mechanisms of action. By humanizing CRBN, the cell line recapitulates human-specific drug responses within a murine cellular context. A key advantage of this system is the combination of human-like pharmacological activity with the experimental versatility of Ba/F3 cells. The Ba/F3 background offers ease of genetic manipulation and robust proliferation, facilitating large-scale screening assays. Meanwhile, humanized CRBN expression ensures clinically relevant readouts, improving the translational predictive value of preclinical findings in targeted degradation therapy development.

Specifications

Catalog Number	KC-5816
Cell Line Name	Ba/F3-mCRBN-humanized-KI-Plus Cell Line
NCBI/UniProt Accession Number	58799/Q8C7D2
Clone Number	2A4
Host Cell Line	Ba/F3
Description	In Ba/F3 cells, the endogenous mouse CRBN gene was replaced with the human CRBN coding sequence under the control of an exogenous promoter.
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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640+10% FBS+8 ng/mL mouse IL-3
Selection Marker	NA
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-mCRBN-humanized-KI-Plus cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of Ba/F3-mCRBN-humanized-KI cell line stable clone using PCR sequencing.

Figure 2: Characterization of Ba/F3-mCRBN-humanized-KI cell line stable clone using western blot.

Cell Resuscitation

Prewarm culture medium (RPMI1640+10% FBS+8 ng/mL mouse IL-3) 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 1:10 every 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

Naruse C, Ishibashi O, Asano M, et al. Differential substrate degradation by super-degron: EGFP in wild-type mouse cells, PD-1 requires CRBN humanization. iScience. 2025; 28(7): 112992. doi: 10.1016/j.isci.2025.112992.
Li SR, Fu J, Wang H, et al. IMiD compounds affect CD34+ cell fate and maturation via CRBN-induced IKZF1 degradation. Blood Adv. 2018;2(5):492–504. doi:10.1182/bloodadvances.2017010348. PMID:29606568; PMCID:PMC5876033.