Abstract

RODRIGO A, Urrego; PAREJA, Andrés; VASQUEZ, Neil A  and  MARQUEZ, María E. The Comet Assay: a technic to evaluate genotoxicity in DNA bovine oocytes. Rev Colom Cienc Pecua [online]. 2005, vol.18, n.3, pp.222-227. ISSN 0120-0690.

The single cell gel electrophoresis or the comet assay is used to measure the DNA damage of individual cells, for most studies the lymphocytes from peripheral blood are used as cellular model. However, during the last years, the application of this technique has been extended to the germinal cells with the purpose of evaluating the integrity of the spermatic DNA, oxidative stress conditions in boar oocytes and bovine embryos besides cellular death in mouse embryos. Applying the comet assay to a system such as the bovine oocytes it becomes indispensable to overcome two big structural barriers in the cumulus-oocyte-complex (COC): first, the degradation of the glycoproteins in the zona pellucida and second, the elimination of the flatten level of the haploid metaphase II bovine oocyte genome. These obstacles do not allow an appropriate migration of the DNA through the agarose gel. In this study it is demonstrated that the use of a lysis solution with proteinase K during 3 h and 10 min in electrophoretic running allows to obtain quantifiable comets of bovine oocytes matured in vitro, which can be used in genotoxic studies.

Keywords : chromatin; DNA strand break; granulose cells; zona pellucida.

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