Cat.No. : CSC-RT2771
Host Cell: HEK293T Target Gene: ATM
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
| Cat. No. | CSC-RT2771 |
| Description | This cell is a stable cell line with a homozygous knockout of human ATM using CRISPR/Cas9. |
| Target Gene | ATM |
| 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 |
DNA damage accumulates with aging. However, whether and how robust DNA repair mechanisms extend lifespan is unclear. Here, researchers demonstrate that the ATM-centric DNA damage response (DDR) declines with aging and age, while low-dose chloroquine (CQ) activates ATM, promotes DNA damage clearance, rescues age-associated metabolic shifts, and extends replicative lifespan. Molecularly, ATM phosphorylates SIRT6 deacetylase, thereby preventing MDM2-mediated ubiquitination and proteasomal degradation. Additional copies of Sirt6 extend lifespan and restore metabolic homeostasis in Atm knockout mice. Furthermore, treatment with CQ significantly extends lifespan in Caenorhabditis elegans but has no effect on ATM-1 mutants. In a mouse model of progeria with low DNA repair capacity, long-term administration of CQ ameliorates premature aging features and extends lifespan. These data highlight a pro-liferative role for ATM and establish a direct causal relationship between robust DNA repair mechanisms and longevity.
In this study, the degradation rate of ectopic and endogenous SIRT6 was greatly increased in ATM knockout HEK293 cells, Atm-/- MEFs, and cells incubated with KU55933 in the presence of cycloheximide (CHX) compared to WT or vehicle controls (Figure 1a-b). The ubiquitination level of FLAG-SIRT6 was significantly increased in ATM knockout cells compared to WT. While the S112A mutant significantly increased the polyubiquitination level of SIRT6, S112D had little effect. In addition, S112A accelerated SIRT6 degradation, while S112D delayed degradation (Figure 1c-d), indicating that phosphorylation of Ser112 by ATM regulates SIRT6 ubiquitination and, thereby, protein stability. In the absence of ATM or SIRT6 S112A mutation, SIRT6's ability to bind to MDM2 was enhanced (Figure 1e). In the search for key residues that are polyubiquitinated by MDM2, the researchers identified two clusters of lysine residues, K143/145 and K346/349. These residues were then KR mutated and it was found that K346/349R significantly reduced the polyubiquitination level of SIRT6. Single KR mutations showed that K346R significantly blocked MDM2-mediated SIRT6 ubiquitination and degradation, while K349R had little effect on it (Figure 1f-g). Collectively, these data indicate that K346 is affected by MDM2-mediated ubiquitination, while ATM-mediated S112 phosphorylation inhibits ubiquitination.
Figure 1. ATM prevents ubiquitination and degradation of SIRT6. (Qian M, 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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