Cat.No. : CSC-RT1847
Host Cell: HEK293T Target Gene: USP7
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
| Cat. No. | CSC-RT1847 |
| Description | HEK293T -USP7(-/-) is a stable cell line with a homozygous knockout of human USP7 using CRISPR/Cas9. |
| Target Gene | USP7 |
| Host Cell | HEK293T |
| Host Cell Species | Homo sapiens (Human) |
| Shipping | 1 vial of knockout cell line |
| Storage | Liquid nitrogen |
| 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 |
Axin is a key scaffolding protein responsible for the formation of the β-catenin destruction complex. The stability of Axin protein is regulated by the ubiquitin-proteasome system, and the regulation of Axin protein cellular concentration has a major impact on Wnt/β-catenin signaling. Here, researchers identified USP7 as a potent negative regulator of Wnt/β-catenin signaling through CRISPR screening. Genetic ablation or pharmacological inhibition of USP7 significantly increased Wnt/β-catenin signaling in multiple cellular systems. USP7 directly interacts with Axin through its TRAF domain and promotes the deubiquitination and stabilization of Axin. Inhibition of USP7 regulates osteoblastic and adipocyte differentiation by increasing Wnt/β-catenin signaling. These studies reveal a key mechanism to prevent excessive degradation of Axin and identify USP7 as a target to sensitize cells to Wnt/β-catenin signaling.
The researchers found that the new generation of USP7 inhibitors Almac430, FT67129, and P5042931 significantly increased STF-GFP in parental HEK293T cells, but not in HEK293T USP7 knockout cells (Figure 1a). Almac4 enhanced STF-Luc activity in HEK293T cells in a dose-dependent manner, with or without exogenous Wnt3a (Figure 1b). Almac4 also enhanced Wnt-induced accumulation of active β-catenin in various cell lines, including HEK293T, MEF, RKO, YAPC, and U2OS (Figures 1c-e). Almac4 consistently increased the expression of β-catenin target genes AXIN2 and LEF1 in YAPC cells (Figure 2f). In addition, the researchers measured the effects of various USP7 inhibitors on Wnt/β-catenin signaling and cell proliferation using the STF-Luc assay and the Cell Titer-Glo (CTG) assay in a dose-response manner (Figure 1g). All new-generation USP7 inhibitors, including Almac430, Almac4732, P5042931, FT67129, and GNE-677633, increased the STF reporter gene. Among all compounds tested, Almac4 and Almac47 had the strongest Wnt-stimulating activity. Both compounds also did not affect the proliferation of HEK293T USP7 knockout cells at all tested concentrations, indicating that Almac4 and Almac47 have minimal off-target activity. These data indicate that pharmacological inhibition of USP7 enhances Wnt/β-catenin signaling in different cell lines.
Figure 1. USP7 inhibitors augment Wnt/β-catenin signaling. (Ji L, et al., 2019)
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.
USP7 knockout cells are cells in which the USP7 gene has been either genetically deleted or disrupted so that it is no longer functional. Good experimental results were obtained.
USP7 knockout cells can be used to investigate the protein's role in other cellular processes, such as DNA damage response, viral replication, and immune system regulation. I recommend Creative Biogene'cell line.
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