Cat.No. : CSC-RT1976
Host Cell: HEK293T Target Gene: CRBN
Size: >1x10^6 cells/vial Validation: Sequencing
| Cat. No. | CSC-RT1976 |
| Description | 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 |
| Host Cell Species | Homo sapiens (Human) |
| Cell Type | Epithelial |
| Size | >1x10^6 cells/vial |
| SequencingResult | 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 |
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)
A: DMEM supplemented with 10% fetal bovine serum.
It is not required to add the selection antibiotics when culturing the KO cells.
A: The knockout cell product is validated by PCR amplification and Sanger Sequencing to confirm the mutation at the genomic level. Please find the detailed mutation info in the datasheet.
A: Single clonal cell.
A: No. This knockout cell product is generated using the CRISPR/Cas9 system to induce small insertions or deletions (indels) resulting in frameshift mutations. Although these frameshift mutations typically disrupt the coding gene, there is a possibility that the non-functional transcript may still be transcribed. Consequently, this could potentially yield misleading results when analyzed by RT-qPCR.
A: The cell line should be stored in liquid nitrogen for long-term preservation.
A: For most cases, we often keep at least 2 clones with different frameshift mutations. Please feel free to contact us to check if there are additional available clones.
If your question is not addressed through these resources, you can fill out the online form below and we will answer your question as soon as possible.
CRBN knockout cells can be invaluable tools for drug discovery. This CRBN knockout cell line is very helpful for our research.
Great buy! By comparing the behavior of CRBN knockout cells to the wild-type cells, we can assess the impact of CRBN loss-of-function on various cellular processes, such as protein degradation, transcriptional regulation, and DNA damage response.
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