Human ATM Knockout Cell Line-HEK293T
Cat.No. : CSC-RT2771
Host Cell: HEK293T Target Gene: ATM
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
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Cell Line Information
Cell Culture Information
Safety and Packaging
| Cat. No. | CSC-RT2771 |
| Cell Line Information | This cell is a stable cell line with a homozygous knockout of human ATM using CRISPR/Cas9. |
| Target Gene | ATM |
| Host Cell | HEK293T |
| Size Form | 1 vial (>10^6 cell/vial) |
| Shipping | Dry ice package |
| Storage | Liquid Nitrogen |
| Species | Human |
| 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 protein encoded by the ataxia-telangiectasia mutated (ATM) gene is essential for maintaining cellular genomic integrity. The ATM protein is a serine/threonine kinase that is activated following DNA double-strand breaks, which are often induced by ionizing radiation and other genotoxic stresses. Once activated, ATM phosphorylates a large number of downstream substrates, including p53, CHK2, BRCA1, NBS1, and many others that synergistically initiate cell cycle arrest and DNA repair mechanisms. The kinase activity of this protein is essential to prevent the propagation of damaged DNA, thereby preventing genomic instability and cellular carcinogenesis.
Mutations in the ATM gene are associated with the autosomal recessive disorder ataxia-telangiectasia (A-T), which is characterized by progressive cerebellar ataxia, telangiectasias, immunodeficiency, and a predisposition to cancer, particularly lymphoid malignancies. In addition to A-T, heterozygous mutations in ATM are associated with an increased risk of various cancers, most notably breast cancer, suggesting that partial loss of ATM function can significantly impact cancer susceptibility. As such, ATM has generated considerable interest in oncology, particularly as a potential biomarker of cancer susceptibility and a target for therapeutic intervention.
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)
Study of DNA Damage Response: ATM is a critical kinase involved in the detection and repair of DNA double-strand breaks. The ATM knockout cell line provides a model to study how cells respond to DNA damage when ATM is absent, offering insights into the mechanisms of DNA repair and genome stability.
Cancer Research: Deficiencies in ATM are associated with cancer predisposition, particularly in ataxia-telangiectasia patients. Using the ATM knockout HEK293T cell line, researchers can investigate how the loss of ATM function contributes to cancer development, progression, and therapeutic resistance, aiding in the identification of potential therapeutic targets.
Drug Screening and Development: This knockout cell line serves as a vital tool for screening anti-cancer drugs and agents that target the DNA damage response pathway. Researchers can assess the efficacy and specificity of new drugs in the absence of ATM, which helps in the development of targeted therapies for ATM-deficient tumors.
Cell Cycle Regulation Research: ATM has a role in the regulation of the cell cycle, particularly at the checkpoints that ensure proper DNA damage repair before cell division. The ATM knockout HEK293T cell line allows scientists to study how the cell cycle is altered without ATM, providing valuable insights into cell cycle control mechanisms.
Genetic Interaction Mapping: ATM interacts with various proteins in the DNA damage response network. The knockout cell line facilitates the mapping of genetic interactions and the identification of other proteins and pathways that compensate for the loss of ATM.
For research use only. Not intended for any clinical use.
