KC-5817

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 characterized by the precise knock-in of key humanizing point mutations into the endogenous mouse Crbn gene. This engineered model is primarily used in the development of targeted protein degradation therapies, especially for the high-throughput screening of molecular glue degraders and the study of their mechanisms of action. The humanization enables the murine cells to mimic the human-specific response to CRBN-targeting drugs, providing a reliable platform for the accurate evaluation of candidate compounds.The outstanding advantage of this model lies in its combination of human-specific drug responsiveness with the experimental convenience of a murine cell system. The Ba/F3 cell background ensures excellent genetic manipulability and proliferation characteristics, making it suitable for large-scale screening experiments. Concurrently, the expression of humanized CRBN guarantees the clinical relevance of drug screening outcomes, significantly enhancing the success rate of translation from preclinical research to clinical applications.

Specifications

Catalog Number	KC-5817
Cell Line Name	Ba/F3-mCRBN-humanized-KI-Plus Cell Line
NCBI/UniProt Accession Number	58799/Q8C7D2
Clone Number	3B2
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.