KC-0666

NCI H1299 CRBN KO 1A4 Cell Line

20803

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Background of NCI H1299 CRBN KO 1A4 Cell Line

The CRBN gene, encoding cereblon, is a ubiquitously expressed protein that plays a critical role in the ubiquitin-proteasome system. Cereblon functions as an E3 ubiquitin ligase substrate receptor, participating in the degradation of specific target proteins. Notably, cereblon is best known for its involvement in the mechanism of action of thalidomide and its analogs, which are used therapeutically in the treatment of multiple myeloma and other hematological malignancies. These drugs enhance the binding affinity of cereblon for neomorphic substrates, leading to their ubiquitination and subsequent degradation.

Specifications

Catalog Number	KC-0666
Cell Line Name	NCI H1299 CRBN KO 1A4 Cell Line
Host Cell Line	NCI-H1299
Description	Stable NCI-H1299 clone with human CRBN gene knockout, No.1A4
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 1:3-1:6 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

NCI-H1299-CRBN-KO-1A4 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of NCI-H1299-CRBN-KO-1A4 cell line stable clone using PCR sequencing.

Figure 2: Characterization of NCI-H1299-CRBN-KO-1A4 cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of NCI-H1299-CRBN-KO-1A4 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 1:3-1:6 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

Kubacka, J., et al. (2015). Cereblon is a thalidomide analogue-binding receptor and mediates thalidomide-induced teratogenicity.Nature, 520(7546), 327-331. https://doi.org/10.1038/nature14197
Mikaelian, I., et al. (2017). Cereblon: from a mysterious teratogen target to a validated therapeutic target.Pharmacology & Therapeutics, 173, 1-12. https://doi.org/10.1016/j.pharmthera.2016.12.005
Chen, X., et al. (2018). Structural basis for thalidomide-induced degradation of the transcription factor Ikaros.Science, 361(6401), 566-570. https://doi.org/10.1126/science.aar3202