Cat.No. : CSC-RT1286
Host Cell: HeLa Target Gene: TNFRSF1A
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
| Cat. No. | CSC-RT1286 |
| Description | A stable cell line with a homozygous knockout of human TNFRSF1A using CRISPR/Cas9. |
| Target Gene | TNFRSF1A |
| Host Cell | HeLa |
| Host Cell Species | Homo sapiens (Human) |
| Shipping | 10^6 cells/tube |
| 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 1:4-1:6. |
| Freeze Medium | Complete medium supplemented with 10% (v/v) DMSO |
| Gene Name | TNFRSF1A |
| Gene ID | 7132 |
| 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 |
Microtubule-targeting agents (MTAs) are one of the most widely used classes of chemotherapeutic drugs, and their mechanism of action has long been assumed to be blocking mitosis in rapidly dividing tumor cells. Contrary to this view, here researchers show that - in many cancer cell types - MTAs act by triggering membrane TNF (memTNF)-mediated cell-to-cell killing, which is very different from other non-MTA cell cycle blockers. Both memTNF and solTNF bind to TNF receptor 1 (TNFR1, also known as tumor necrosis factor receptor superfamily member 1A (TNFRSF1A)), thereby activating downstream cell death pathways. In cells that do not express RIP3 or have defective RIP3 signaling, TNFR1 activates the initiator protease caspase-8, which subsequently cleaves and activates the downstream executioner proteases caspase-3/7, leading to apoptosis. Here, researchers uncover a highly specific feature of the MTA family of drugs that distinguishes them from other mitotic blockers: both microtubule depolymerizers and stabilizers can directly induce TNF signaling-mediated tumor cell death, either by apoptosis in RIP3-deficient cancer cells or by necroptosis in RIP3-expressing cancer cells. Surprisingly, this MTA-induced cancer cell death was dependent on JNK/c-Jun-regulated accumulation of membrane TNF (memTNF) but not solTNF, through which it induces cancer cell killing by memTNF.
Most human cancer cells, such as HeLa cells, are resistant to necroptosis but sensitive to apoptosis due to the lack of expression of the necroptosis-essential gene RIP3. Here, the researchers tested the cell death response of human cancer cell lines lacking RIP3 to MTA. The study found that MTA also induced TNF-dependent apoptosis in HeLa cells. Blocking soluble TNF shedding by inhibiting TACE activity did not interfere with MTA-induced apoptosis; when TNFR1 was knocked out, cell death was abolished (Figure 1a-c). Ectopic expression of TNFR1 in TNFR1 (TNFRSF1A) knockout HeLa cells restored the apoptotic response to MTA (Figure 4d). The researchers next confirmed that MTA induced apoptosis in a variety of human cancer cell lines lacking RIP3 (Figure 1e-g). In fact, the mRNA levels of JUN and membrane-bound TNF in these human cancer cells were also upregulated by MTA treatment (Figure 1h,i).
Figure 1. MTAs induce memTNF-mediated apoptosis in RIP3-deficient human carcinoma cell lines. (Zhang, Jing, et al. 2020)
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.
As a researcher focused on cytokine signaling, the Human TNFRSF1A Knockout Cell Line-HeLa has been invaluable. The clear absence of TNFRSF1A expression allows for meticulous analysis of downstream effects without any background noise.
The precision and reliability of this cell line in simulating TNFRSF1A knockouts have accelerated our understanding of inflammatory response mechanisms. The quality of the knockout has always been on point, saving us a lot of troubleshooting time and allowing us to focus on data analysis and interpretation.
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