USP7 Knockout Cell Line-HEK293T
Cat.No. : CSC-RT1847
Host Cell: HEK293T Target Gene: USP7
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
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Cell Line Information
Cell Culture Information
Safety and Packaging
| Cat. No. | CSC-RT1847 |
| Cell Line Information | HEK293T -USP7(-/-) is a stable cell line with a homozygous knockout of human USP7 using CRISPR/Cas9. |
| Target Gene | USP7 |
| Host Cell | HEK293T |
| Shipping | 1 vial of knockout cell line |
| Storage | Liquid nitrogen |
| Species | Human |
| Gene Symbol | USP7 |
| Gene ID | 7874 |
| 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
USP7, also known as ubiquitin-specific peptidase 7, is a gene encoding a deubiquitinating enzyme that plays a crucial role in various cellular processes. USP7 is widely expressed in human tissues and has been found to be integral in regulating various cellular pathways, including DNA damage response, regulation of gene expression, and immune responses. One of its most intensively studied roles is in the p53 signaling pathway. USP7 can deubiquitinate and stabilize the tumor suppressor protein p53, thereby exerting anticancer effects. Paradoxically, USP7 has also been shown to stabilize MDM2, an E3 ubiquitin ligase that targets p53 for degradation. This dual role highlights the complex regulatory network in which USP7 participates and underscores its importance in maintaining cellular homeostasis.
In addition to the p53 pathway, USP7 is also involved in the regulation of the Wnt signaling pathway, NF-κB signaling, and chromatin dynamics through interactions with various substrates such as FOXO4, PTEN, and DNMT1. These interactions suggest that USP7 is a multifunctional enzyme with multiple functions that contribute to processes such as apoptosis, cell proliferation, and genomic stability.
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)
Here are some potential applications for USP7 Knockout Cell Line-HEK293T:
Cancer research: By understanding the role of USP7 in cell proliferation and apoptosis, researchers can identify potential therapeutic targets for cancer treatment, as USP7 is known to stabilize oncogenic proteins and inhibit tumor suppressor pathways.
Drug screening: By evaluating compounds in a USP7-deficient setting, scientists can identify drugs that are particularly effective against cells lacking this deubiquitinase, potentially enabling targeted cancer therapies with fewer side effects.
Genomic studies: USP7 knockout provides a unique tool for genomic studies aimed at elucidating the complex regulatory networks involved in ubiquitination. Understanding how the loss of USP7 affects gene expression and protein stability helps map out key pathways that could potentially be used for therapeutic intervention.
Cell cycle regulation: USP7 plays an important role in cell cycle regulation. Knockout cell lines facilitate the study of the effects of USP7 loss on cell cycle checkpoints, DNA repair mechanisms, and overall cell health, contributing to a broader understanding of cell cycle dynamics and cancer biology.
Epigenetics and chromatin dynamics: USP7 has been implicated in the regulation of chromatin remodeling and epigenetic modifications. Using USP7 knockout HEK293T cells, researchers can study how loss of USP7 affects chromatin state, histone modifications, and thus gene expression and cell differentiation pathways.
Protein-protein interaction studies: As a deubiquitinating enzyme, USP7 interacts with a large number of proteins. The knockout model provides a simplified system to dissect these interactions and understand the impact of USP7 loss on protein networks.
For research use only. Not intended for any clinical use.
