Utilising the comet assay for the detection of mouse models in a large scale genetic screen to determine genetic components of radiation sensitivity

C. A. Schindewolf a, A. Hartmannb, H.V. Liebscherc, W. Frieauffb, H. Fuchsa, E. Binderd, F. Ohld, D. Soewartoa, M. D. Gomolkae, S. Hornhardte, T. Junge, M. M. Hrabé de Angelisa

aInstitute for Experimental Genetics, GSF Research Centre for Health and Environment, Ingolstädter Landstr. 1, D-85764 Neuherberg, Germany, b Novartis Pharma AG, Preclinical Safety, WSH-2881, CH-4002 Basel, Switzerland, cInstitute for Biomathematics and Biometry, GSF Research Centre for Health and Environment, Ingolstädter Landstr. 1, D-85764 Neuherberg, Germany, dMax-Planck-Institute of Psychiatry, Kraepelinstr. 2, D-80804 Munich, Germany, eInstitute for Radiation Hygiene, Federal Office of Radiation Protection, Ingolstädter Landstr. 1, D-5764 Neuherberg, Germany

Aim: Patients in radiation therapy, who are heterozygote for genetic diseases such as Ataxia telangiectasia (ATM), Nijmegen Breakage Syndrome and Fanconi Anemia, have a known predisposition for radiation sensitivity which is often accompanied by an increased risk of tumour development. Such patients are either affected by mutations in their DNA repair system or, as the effectiveness of the DNA repair system is itself dependent upon other cellular processes, by genetic aberrations in the regulation of the cell cycle, signal transduction, DNA replication or DNA recombination processes. Unfortunately, it is difficult to identify these genetic deficiencies prior to exposure to ionising radiation. For this reason, animal (e.g. mouse) models which display these genetic deficiencies are essential. In the framework of a large ENU mutagenesis screen for mutant mice, we searched for mice with a reduced capacity for DNA repair. Methods: To search within a large scale genetic screen for genetic components of radiation sensitivity, it is necessary to establish an automated ”in vitro” assay. We utilized the alkaline single cell gel electrophoresis (Comet assay) on lymphocytes from mice to detect those mice which display an altered reaction of DNA repair capacity. A fully automated image analysis system for the analysis of Comet assay slides was utilized, along with laboratory adaptations towards automation. Results: After irradiation, we detected a mouse mutant displaying a reduced capacity for DNA repair. The mouse is smaller in stature than normal, hyperactive, has body tremors and is unable to reproduce. An inherent inability to produce sperm appears to be partially responsible for the phenotype as no sperm were found upon dissection. Conclusions: We utilized the alkaline Comet assay to monitor a mutant mouse population and detect mice with a reduced DNA repair capacity. Our results indicate that it is possible to detect individuals with a genetic predisposition to radiation sensitivity within a normal population.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Predictive Markers.