KC-6504

NCI-H1975-CRBN-EGFR-KO-EGFR-L858R-C797S-Flag Cell Line

63138

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Background of NCI-H1975-CRBN-EGFR-KO-EGFR-L858R-C797S-Flag 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-6504
Cell Line Name	NCI-H1975-CRBN-EGFR-KO-EGFR-L858R-C797S-Flag Cell Line
Clone Number	1A3
Host Cell Line	NCI-H1975-EGFR-KO-EGFR-L858R-C797S-Flag
Description	Stable NCI-H1975-EGFR-KO-EGFR-L858R-C797S-Flag clone with human CRBN gene knockout
Quantity	One vial 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+1μg/ml puromycin
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

Cell Line Generation

NCI-H1975-CRBN-EGFR-KO-EGFR-L858R-C797S-Flag cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of NCI-H1975-CRBN-EGFR-KO-EGFR-L858R-C797S-Flag cell line stable clone using PCR sequencing.

Figure 2: Characterization of NCI-H1975-CRBN-EGFR-KO-EGFR-L858R-C797S-Flag cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of NCI-H1975-CRBN-EGFR-KO-EGFR-L858R-C797S-Flag cell line stable clone using western blot.

Cell Resuscitation

Prewarm culture medium (RPMI1640+10%FBS+1μg/ml puromycin)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.

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.