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Ku80-/- MEF (Mouse Embryonic Fibroblast) cells are genetically engineered fibroblast cells derived from mice that lack the Ku80 gene (XRCC5). The Ku80 protein, in conjunction with Ku70, forms the Ku heterodimer, which is essential for the non-homologous end joining (NHEJ) pathway of DNA double-strand break (DSB) repair. The absence of Ku80 in these cells impairs their ability to effectively repair DSBs, making them a valuable model for studying the role of the NHEJ pathway in genomic stability, DNA repair mechanisms, and cancer biology.
Ku80-/- MEF cells exhibit increased sensitivity to ionizing radiation and other DNA-damaging agents due to their compromised DSB repair capacity. These cells also tend to accumulate chromosomal aberrations and exhibit genomic instability. The lack of Ku80 affects not only DNA repair but also other cellular processes such as V(D)J recombination, which is crucial for the development of a diverse repertoire of antibodies and T-cell receptors in the immune system.
Research using Ku80-/- MEF cells has provided significant insights into the molecular mechanisms of NHEJ and the broader implications of defective DNA repair. These studies are crucial for understanding the development of cancer and other diseases associated with genomic instability. Additionally, they help in the exploration of potential therapeutic targets for enhancing DNA repair in cancer cells, thereby improving the efficacy of cancer treatments that rely on inducing DNA damage in tumor cells.
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- Viruses: Transformant: Simian virus 40 (SV40)
- Mutational profile: Mutation: Ku80-/-
- cultureMedium: DMEM, w: 4.5 g/L Glucose, w: 4 mM L-Glutamine, w: 3.7 g/L NaHCO3, w: 1.0 mM Sodium pyruvate (Cytion article number 820300a)
- supplements: Supplement the medium with 10% FBS
- dissociationReagent: Accutase
- subculturing: Remove the old medium from the adherent cells and wash them with PBS that lacks calcium and magnesium. For T25 flasks, use 3-5 ml of PBS, and for T75 flasks, use 5-10 ml. Then, cover the cells completely with Accutase, using 1-2 ml for T25 flasks and 2.5 ml for T75 flasks. Let the cells incubate at room temperature for 8-10 minutes to detach them. After incubation, gently mix the cells with 10 ml of medium to resuspend them, then centrifuge at 300xg for 3 minutes. Discard the supernatant, resuspend the cells in fresh medium, and transfer them into new flasks that already contain fresh medium.
- freezeMedium: As a cryopreservation medium, use complete growth medium (including FBS) + 10% DMSO for adequate post-thaw viability, or CM-1 (Cytion catalog number 800100), which includes optimized osmoprotectants and metabolic stabilizers to enhance recovery and reduce cryo-induced stress.
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