Human CRBN Knockout Cell Line-A549
Cat.No. : CSC-RT2717
Host Cell: A549 Target Gene: CRBN
Size: 2x10^6 cells/vial Validation: Sequencing
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Cell Line Information
Cell Culture Information
Safety and Packaging
| Cat. No. | CSC-RT2717 |
| 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 | A549 |
| Cell Type | Epithelial |
| Size | 2x10^6 cells/vial |
| Sequencing Result | Allele-1: 17 bp deletion in exon Allele-2: 10 bp deletion in exon |
| 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:F12 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
Cereblon (CRBN) is a gene that encodes a protein of the same name. The gene is located on human chromosome 3 and plays an important role in regulating protein degradation and is evolutionarily conserved across species from plants to humans. The CRBN protein is a core component of an E3 ubiquitin ligase complex that includes damaged DNA-binding protein 1 (DDB1), Cullin-4A (CUL4A), and regulator of cullins 1 (ROC1). This complex is essential for tagging proteins with ubiquitin to mark them for proteasomal degradation, a key process in maintaining cellular homeostasis.
The CRBN gene is essential for various developmental processes, including limb and auditory vesicle formation. Through its role in the E3 ubiquitin ligase complex, CRBN helps regulate the levels of fibroblast growth factors such as FGF8 and FGF10, which are essential for normal embryonic development. In the absence of cereblon, the DDB1 protein translocates and forms a complex with DDB2, which is involved in DNA damage repair, suggesting competitive binding and multifunctional roles for these proteins. In addition to its developmental role, CRBN is involved in regulating potassium channels, particularly the large conductance calcium-activated potassium channel (KCNMA1).
Cereblon (CRBN) has been identified as a major target for immunomodulatory drugs in multiple myeloma. Here, researchers demonstrate for the first time that CRBN expression plays a functional role in lung cancer progression by regulating autophagy through Toll-like receptors (TLR)2, TLR4, and TLR7. TLR signaling has been implicated in the induction of autophagy and plays a key role in lung cancer progression and pathogenesis. These studies suggest that CRBN could serve as an effective prognostic marker for lung cancer and provide important implications for clinical and translational lung cancer biology.
In this study, cell migration and invasion were significantly enhanced in CRBN knockout (CRBNKO) A549 cells under TLR2 (HKLM), TLR4 (LPS), or TLR7 (IQM) stimulation. Significant inhibition was observed in the presence of autophagy inhibitors 3-MA or CQ. Importantly, LC3-II levels and LC3 dots, representing autophagy induction, were significantly enhanced in CRBNKO A549 treated with TLR agonists (Figure 1A-C, LC3-II levels; Figure 1D,E, LC3 puncta). TLR4 signaling induces autophagy through BECN1 ubiquitination by TRAF6. BECN1 ubiquitination was significantly increased in CRBNKO A549 cells treated with HKLM, LPS, or IQM compared with Ctrl A549 cells (Figure 1F). The production of IL-6, CCL2, CCL20, and MMP2 is required for enhanced migration and invasion of lung cancer cells after TLR activation. In addition, CRBNKO A549 cells significantly increased IL-6, CCL2, CCL20, and MMP2 in response to three TLR agonists compared with Ctrl A549 (Figure 1G-J), while significant inhibition was observed upon co-treatment with autophagy inhibitors (Figure 1G-J). Finally, the researchers found that CRBNKO A549 cells treated with TLR agonists significantly increased single cell migration rate and colony number compared with Ctrl A549 cells. In summary, CRBN is downregulated in lung cancer cells and is associated with lung cancer progression. These studies demonstrate the association between TLR stimulation and gene signatures associated with lung cancer progression and autophagy.
Figure 1. TLR2, TLR4 and TLR7 enhance autophagy and cancer‐promoting cytokines in CRBN knockout (CRBNKO) A549 lung cancer cells. (Kim M J, et al., 2022)
1. Drug Discovery and Development: The human CRBN knockout cell line - A549 is particularly valuable in the field of drug discovery and development. Researchers can use this cell line to study the effects of new drug compounds on cells that lack the CRBN gene. This helps identify potential targets for drugs that may treat diseases associated with dysfunction or loss of the CRBN protein.
2. Cancer Research: A549 is a widely used human lung cancer cell line, and knocking out the CRBN gene in these cells can help researchers understand its role in cancer progression. By comparing the growth, survival, and response to treatment of CRBN knockout cells to wild-type cells, scientists can elucidate the pathways by which CRBN affects the behavior of cancer cells.
3. Protein Degradation Mechanism Studies: Cereblon is an essential component of the E3 ubiquitin ligase complex, which is involved in protein degradation via the ubiquitin-proteasome system. CRBN knockout A549 cells can be used to study the specific substrates that are targeted by CRBN and the consequences of the accumulation of these substrates when CRBN is missing. This can provide insights into a variety of diseases that may be associated with defects in protein degradation.
4. Studying Neurological Diseases: Researchers can use CRBN knockout A549 cells in co-culture systems or as models to understand how CRBN deficiency indirectly affects neuronal cells, which may lead to new approaches to treat neurological diseases.
For research use only. Not intended for any clinical use.
