Cat.No. : CSC-RT2740
Host Cell: HEK293T Target Gene: OGG1
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
| Cat. No. | CSC-RT2740 |
| Description | This cell is a stable cell line with a homozygous knockout of human OGG1 using CRISPR/Cas9. |
| Target Gene | OGG1 |
| Host Cell | HEK293T |
| Host Cell Species | Homo sapiens (Human) |
| Size Form | 1 vial (>10^6 cell/vial) |
| Shipping | Dry ice package |
| 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 |
The onset of inflammation is associated with reactive oxygen species and oxidative damage to macromolecules such as 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA. Since 8-oxoguanine DNA glycosylase 1 (OGG1) binds to 8-oxoG and Ogg1-deficient mice are resistant to acute and systemic inflammation, the researchers hypothesized that OGG1 inhibition may represent a strategy to prevent and treat inflammation. They developed TH5487, a selective OGG1 active site inhibitor that blocks OGG1 binding to and repair of 8-oxoG and is well tolerated by mice. TH5487 prevents tumor necrosis factor α-induced OGG1-DNA interactions at guanine-rich pro-inflammatory gene promoters. This in turn reduces nuclear factor κB DNA occupancy and pro-inflammatory gene expression, leading to reduced immune cell recruitment in the lungs of mice. Thus, they present a proof of concept that targeting oxidative DNA repair could alleviate inflammatory conditions in vivo.
For OGG1 inhibitors to exert pharmacological effects, they need to bind to OGG1 in cells and inhibit its activity. TH5487 increased the melting temperature of OGG1 in human cells (Figure 1A), indicating that TH5487 binds to its target in living cells and protects it from thermal denaturation. In addition, TH5487 also impaired KBrO3-induced genomic 8-oxoG repair. TH5487 caused a significant increase in genomic 8-oxoG after 2.5 hours (Figure 1B and C), and after 24 hours, 50 ± 8% of 8-oxoG remained in TH5487-treated cells (Figure 1C) without disrupting proliferation. Therefore, both genomic 8-oxoG and TH5487 are well tolerated by cells. Treatment with TH5487 increased the nuclear mobility of OGG1-GFP at 3 and 5 h after KBrO3 exposure (Figure 1D and E), indicating that TH5487 prevents OGG1 from binding to its genomic substrates in living cells. In addition, OGG1 knockout HEK293T cell showed reduced induction of CXCL1 [chemokine (C-X-C motif) ligand 1] mRNA after tumor necrosis factor-α (TNFα) stimulation (Figure 1F). Treatment with 5 μM TH5487 reduced CXCL1 expression by more than 50% in wild-type cells but not in OGG1 knockout HEK293T cells (Figure 1F). Therefore, this compound can be used to specifically inhibit OGG1-dependent proinflammatory gene expression.
Figure 1. TH5487 engages OGG1 in cells, inhibits DNA repair, and alters OGG1 chromatin dynamics. (Visnes T, et al., 2018)
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.
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By studying OGG1 knockout cells, we can observe the consequences of impaired OGG1 function and gain valuable insights into the mechanism and importance of OGG1-mediated DNA repair. Good experimental results were obtained.
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