CRBN Knockout Cell Line-HEK293T
Cat.No. : CSC-RT1976
Host Cell: HEK293T Target Gene: CRBN
Size: >1x10^6 cells/vial Validation: Sequencing
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Cell Line Information
Cell Culture Information
Safety and Packaging
| Cat. No. | CSC-RT1976 |
| Cell Line Information | This cell line is a stable cell line with a homozygous knockout of human CRBN using CRISPR/Cas9. |
| Target Gene | CRBN |
| Gene ID | 51185 |
| Genotype | CRBN (-/-) |
| Host Cell | HEK293T |
| Cell Type | Epithelial |
| Size | >1x10^6 cells/vial |
| Sequencing Result | Homozygous: Exon1 deletion |
| Revival | Rapidly thaw cells in a 37°C water bath. Transfer contents into a tube containing pre-warmed media. Centrifuge cells and seed into a 25 cm2 flask containing pre-warmed media. |
| Media Type | Cells were cultured in DMEM supplemented with 10% fetal bovine serum. |
| Growth Properties | Cells are cultured as a monolayer at 37°C in a humidified atmosphere with 5% CO2. Split at 80-90% confluence, approximately 1:3-1:6. |
| Freeze Medium | Complete medium supplemented with 10% (v/v) DMSO |
| Mycoplasma | Negative |
| Format | One frozen vial containing millions of cells |
| Storage | Liquid nitrogen |
| Safety Considerations |
The following safety precautions should be observed. 1. Use pipette aids to prevent ingestion and keep aerosols down to a minimum. 2. No eating, drinking or smoking while handling the stable line. 3. Wash hands after handling the stable line and before leaving the lab. 4. Decontaminate work surface with disinfectant or 70% ethanol before and after working with stable cells. 5. All waste should be considered hazardous. 6. Dispose of all liquid waste after each experiment and treat with bleach. |
| Ship | Dry ice |
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Background
Case Study
Applications
The CRBN (cereblon) gene is located on human chromosome 3 and encodes a protein that is an essential component of the CRL4-CRBN E3 ubiquitin ligase complex. This protein complex is essential for targeting specific substrate proteins for degradation by the ubiquitin-proteasome system. By tagging these substrates with ubiquitin molecules, CRL4-CRBN designates them for degradation, thereby controlling protein levels within the cell and promoting a variety of cellular processes, including cell cycle regulation, DNA repair, and responses to oxidative stress.
Mutations or dysregulation of the CRBN gene can lead to a variety of diseases and conditions. For example, loss-of-function mutations in CRBN are associated with autosomal recessive nonsyndromic intellectual disability, highlighting the important role of this gene in neurodevelopment and cognitive function. In addition, alterations in CRBN expression or function can affect the efficacy of thalidomide and its derivatives, called immunomodulatory drugs (IMiDs), in cancer treatment, making CRBN an important focus of oncology research. Once notorious for causing severe birth defects, thalidomide is now used to treat certain blood cancers due to its immunomodulatory and anti-inflammatory properties. The study found that thalidomide binding to CRBN alters the substrate specificity of the CRL4-CRBN complex, leading to the degradation of new target proteins, including Ikaros and Aiolos, which are transcription factors implicated in the pathophysiology of multiple myeloma.
The immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide are approved drugs for the treatment of multiple myeloma. IMiDs induce cereblon (CRBN) E3 ubiquitin ligase-mediated ubiquitination and degradation of Ikaros transcription factors Ikaros (IKZF1) and Aiolos (IKZF3), which are essential for multiple myeloma. Studies here show that expression of human CRBN or the CrbnI391V mutant abrogates IMiD-induced degradation of IKZF1 and IKZF3 in mouse MOPC.315.BM.Luc.eGFP and 5T33MM multiple myeloma cells. Consequently, lenalidomide and pomalidomide reduced cell viability of mouse multiple myeloma cells expressing CrbnI391V in vitro in a dose-dependent manner. The sensitivity of mouse cells expressing CrbnI391V to IMiDs was highly correlated with their dependence on IKZF1. After transplantation, MOPC.315.BM.Luc.eGFP cells expressing mouse CrbnI391V induced multiple myeloma in mice, and treatment with lenalidomide and pomalidomide significantly delayed tumor growth.
In this study, to confirm that IMiD-mediated degradation of IKZF1 and IKZF3 was due to the restoration of new substrate binding to CRBN, immunoprecipitation was performed in CRBN knockout HEK293T cells transfected with HA-IKZF3 and FLAG-tagged hCRBN, mCrbn, or mCrbnI391V after lenalidomide treatment. The study found that HA-IKZF3 bound to FLAG-tagged hCRBN and mCRBNI391V, but not to mCRBN, upon lenalidomide treatment.
Figure 1. Expression of hCRBN or mCrbnI391V restores neo-substrate binding to CRBN upon lenalidomide treatment. HA-tagged IKZF3 was immunoprecipitated in CRBN knockout HEK293T cells expressing FLAG-tagged hCRBN, mCrbn, or mCrbnI391V. Cells were treated with lenalidomide for two hours and the interaction of HA-IKZF3 with CRBN was detected. (Röhner L, et al., 2021)
CRBN (cereblon) is a protein that plays a key role in the ubiquitin-proteasome system and is a key part of the CRL4CRBN E3 ubiquitin ligase complex. The following are some important applications of CRBN Knockout Cell Line-HEK293T:
Drug Development: These cells can be used as preclinical models to evaluate the efficacy and off-target effects of CRBN-modulating drugs, such as immunomodulatory drugs (IMiDs) such as lenalidomide, helping to accelerate the drug discovery and validation process.
Functional Genomics: CRBN knockout in HEK293T cells facilitates high-throughput screening to identify genes and pathways that interact with or compensate for CRBN loss, providing insights into new therapeutic targets.
Neurodegenerative Disease Research: Given the involvement of CRBN in neuroprotection, these cell lines are valuable for studying the pathophysiology of neurodegenerative diseases, potentially leading to the development of new therapeutic strategies.
Cancer Research: By studying CRBN knockout HEK293T cells, researchers can investigate the contribution of CRBN to tumor growth and survival, aiding the understanding of cancer biology and the development of targeted therapies.
Protein Degradation Pathways: This knockout model allows examination of the role of CRBN in proteasome-mediated protein degradation, aiding a broader understanding of protein quality control mechanisms within cells.
For research use only. Not intended for any clinical use.
