KC-3795

A549-CRBN-KO Cell Line

34792

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Background of A549-CRBN-KO Cell Line

Cereblon is a protein that in humans is encoded by the CRBN gene. The gene that encodes the cereblon protein is found on the human chromosome 3, on the short arm at position p26.3 from base pair 3,190,676 to base pair 3,221,394. CRBN orthologs are highly conserved from plants to humans. Cereblon forms an E3 ubiquitin ligase complex with damaged DNA binding protein 1 (DDB1), Cullin-4A (CUL4A), and regulator of cullins 1 (ROC1).

Specifications

Catalog Number	KC-3795
Cell Line Name	A549-CRBN-KO Cell Line
Clone Number	3A4
Host Cell Line	A549
Description	Stable A549 cell line with endogenous CRBN gene knockout.
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% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS
Selection Marker	NA
Morphology	Epithelial
Subculture	Split saturated culture 1:5-1:10 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

A549-CRBN-KO-3A4 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of A549-CRBN-KO cell line stable clone using PCR sequencing.

Figure 2: Characterization of A549-CRBN-KO cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of A549-CRBN-KO cell line stable clone using western blot.

Cell Resuscitation

Prewarm culture medium (DMEM + 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 1:5-1:10 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 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

Higgins JJ, Pucilowska J, Lombardi RQ, Rooney JP. A mutation in a novel ATP - dependent Lon protease gene in a kindred with mild mental retardation. Neurology. 2004 Nov;63(10):1927–1931.doi:10.1212/01.wnl.0000146196.01316.a2. PMID: 15557513; PMCID: PMC1201536.
Angers S, Li T, Yi X, MacCoss MJ, Moon RT, Zheng N. Molecular architecture and assembly of the DDB1 - CUL4A ubiquitin ligase machinery. Nature. 2006 Oct;443(7111):590–593. doi:10.1038/nature05175. PMID 16964244.