TP53 Knockout Cell Line-293T
Cat.No. : CSC-RT0369
Host Cell: HEK293T Target Gene: TP53
Size: 1x10^6 cells/vial, 1 mL Validation: Sequencing
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Cell Line Information
Cell Culture Information
Safety and Packaging
| Cat. No. | CSC-RT0369 |
| Cell Line Information | This cell line is a stable cell line with a homozygous knockout of human TP53 using CRISPR/Cas9. |
| Target Gene | TP53 |
| Gene ID | 7157 |
| Genotype | TP53 (-/-) |
| Host Cell | HEK293T |
| Cell Type | Epithelial |
| Size | 1x10^6 cells/vial, 1 mL |
| Sequencing Result | Homozygous: 2 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 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 TP53 gene is an important tumor suppressor gene in cells. It is located on human chromosome 17 and encodes a protein called p53. The TP53 gene is one of the genes responsible for controlling cell growth in cells. As a "guard" to maintain the stability and integrity of cell genes, the most important role of the p53 protein is to ensure that cells stop growing or die at the right time (a process called apoptosis or programmed cell death) to prevent cells from becoming cancerous. When the TP53 gene mutates, if the cell has DNA damage, the mutated TP53 gene may not be able to respond through the normal p53 signaling pathway, which may cause these damaged cells to grow and divide uncontrollably.
In addition, TP53 mutations may also directly promote cancer cells. Under normal circumstances, the p53 protein does not stay in the human body, but is rapidly degraded after binding to an enzyme called "MDM2". However, the mutant p53 protein produced after the TP53 mutation cannot bind to MDM2 and degrade and accumulates in the cell. This mutant protein is a tumor-promoting factor. On the one hand, it plays a "gain of function" role in promoting cancer progression or drug resistance. On the other hand, it also induces the body's autoimmune response, produces p53 autoantibodies, and further destroys the normal function of the p53 protein. According to studies, about 50% of malignant tumors have TP53 mutations, which is the most common mutated gene in tumors. Among them, TP53 mutations are found in 96% of ovarian cancers, 54% of invasive breast cancers, 86% of small cell lung cancers, and 75% of pancreatic cancers.
Ewing sarcoma is a pediatric cancer driven by the EWS-ETS transcription factor fusion oncoprotein, which has a stable genomic background. Most tumors express wild-type TP53, so therapies targeting the p53 pathway will benefit most patients. To discover specific targets against TP53 wild-type Ewing sarcoma, researchers used a genome-scale CRISPR-Cas9 screen and identified and validated MDM2, MDM4, USP7, and PPM1D as druggable dependencies. ATSP-7041, a stapled peptide inhibitor of MDM2 and MDM4, showed antitumor efficacy in vitro and in multiple mouse models. P5091, a USP7 inhibitor, and GSK2830371, a Wip1/PPM1D inhibitor, reduced the viability of Ewing sarcoma cells. Combinations of ATSP-7041 with P5091, GSK2830371, and chemotherapeutic agents showed synergistic effects on the p53 pathway. Simultaneous knockout of TP53 rescued the effects of inhibitors including the specific USP7 inhibitor XL-188, highlighting the importance of intact p53 for the observed cytotoxic activity.
Three TP53 wild-type cell lines were infected with CRISPR-Cas9 constructs targeting TP53, and loss of TP53 was demonstrated by a reduction in the increase in p53 protein levels after etoposide treatment (Figure 1 A). Treatment of TP53 knockout cells showed that loss of TP53 completely rescued the cytotoxic effects of ATSP-7041, indicating that the drug has on-target activity (Figure 1 B) and that the response to MDM2/MDM4 inhibition is dependent on intact p53. Similarly, the Wip1 inhibitor GSK2830371 was less effective in TP53 knockout cells than in control cells, suggesting that GSK2830371 has on-target activity (Figure 1 C). However, TP53 knockout did not protect cells from the effects of P5091, suggesting a p53-independent mechanism or that this molecule may have off-target effects (Figure 1 D). Simultaneous loss of TP53 effectively rescued the cytotoxic effects of USP7 knockout, a phenomenon also observed with PPM1D knockout ( Figure 1 , E–G). XL-188 primarily reduced viability in TP53 wild-type Ewing sarcoma cell lines, with particularly strong effects observed in TC32 cells ( Figure 1 H). Surprisingly, TP53 knockout completely reversed the cytotoxic effects of XL-188 ( Figure 1 I), supporting the requirement for functional p53 to be required for the USP7 inhibition response observed in Ewing sarcoma.
Figure 1. Loss of PPM1D and USP7 is rescued by concurrent TP53 loss. (Stolte, Björn, et al. 2018)
The TP53 gene is best known for encoding the key tumor suppressor protein p53, which plays a crucial role in mediating the cellular response to DNA damage and oncogenic stress. Here, we elucidate several key applications of the TP53 knockout 293T cell line.
Cancer Research and Drug Development: The TP53 knockout 293T cell line can be used to study mechanisms of tumorigenesis, delineate pathways by which p53 loss leads to cancer progression, and screen for therapeutics against TP53-deficient tumors. Researchers can gain insights into mechanisms of sensitivity or resistance to various chemotherapeutic agents and develop customized therapies for cancers with TP53 dysfunction.
DNA Damage Response Studies: P53 is an integral part of the DNA damage response system. The TP53 knockout 293T cell line is a powerful tool to study how cells respond to DNA damage in the absence of functional p53. This can include studies of DNA repair pathways, replication stress, and genomic instability.
Cell Cycle Regulation: P53 is a key regulator of the cell cycle, particularly at the G1/S checkpoint. Using the TP53 knockout 293T cell line, researchers can analyze the effects of p53 deficiency on cell cycle dynamics. This can help identify compensatory mechanisms and potential regulatory checkpoints that could be used therapeutically to control the unregulated cell proliferation seen in many cancers.
Apoptosis and Senescence Studies: By utilizing the TP53 knockout 293T cell line, scientists can study the effects of p53 loss on apoptotic pathways and senescence.
Gene Therapy and Synthetic Biology: The TP53 knockout 293T cell line is an excellent model for testing gene therapy techniques designed to restore p53 function or compensate for its loss. Additionally, synthetic biology applications could benefit from such cell lines to engineer new gene circuits or pathways that can function independently or compensatorily in the absence of p53.
For research use only. Not intended for any clinical use.
