KC-6227

TMD8-CRBN-KO Cell Line

62367

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Background of TMD8-CRBN-KO Cell Line

Cereblon (CRBN) is a substrate receptor of the Cullin-4 RING E3 ubiquitin ligase complex (CRL4CRBN) that plays a pivotal role in targeted protein degradation . Originally associated with autosomal recessive non-syndromic mental retardation, CRBN gained clinical prominence as the primary molecular target of thalidomide and its immunomodulatory drugs (IMiDs) derivatives, lenalidomide and pomalidomide . Upon IMiD binding, CRBN undergoes a neomorphic functional shift, recruiting lymphoid transcription factors IKZF1 (Ikaros) and IKZF3 (Aiolos) for ubiquitination and proteasomal degradation, thereby exerting anti-myeloma effects and modulating immune responses . CRBN mutations, including point mutations and splice variants, occur in 12-30% of IMiD-refractory multiple myeloma patients and represent a key mechanism of acquired drug resistance . Beyond hematologic malignancies, CRBN has emerged as a versatile platform for proteolysis-targeting chimeras (PROTACs), enabling targeted degradation of oncogenic proteins in both hematologic and solid tumors . Recent structural and functional studies classify CRBN mutations into loss-of-function, neutral, and agent-dependent categories, guiding therapeutic strategies including next-generation CELMoD agents .

Specifications

Catalog Number	KC-6227
Cell Line Name	TMD8-CRBN-KO Cell Line
Clone Number	1D1
Host Cell Line	TMD8
Description	Stable TMD8 clone with human 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	RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10% FBS
Selection Marker	N/A
Morphology	Lymphoblast
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

Cell Line Generation

TMD8-CRBN-KO cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of TMD8-CRBN-KO cell line stable clone using PCR sequencing.

Figure 2: Characterization of TMD8-CRBN-KO cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of TMD8-CRBN-KO 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% 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

Ito T, et al. (2010). "Identification of a primary target of thalidomide teratogenicity." Science, 327(5971), 1345-1350.
Kortüm KM, et al. (2016). "Targeted sequencing of refractory myeloma reveals a high incidence of mutations in CRBN and Ras pathway genes." Blood, 128(9), 1226-1233.
Thapa R, et al. (2024). "CRBN-PROTACs in Cancer Therapy: From Mechanistic Insights to Clinical Applications." Chem Biol Drug Des, 104(5), e70009.