Quantification of DNA damage by q-PCR in cryopreserved zebrafish Primordial Germ Cells


Author's address: Vanesa Robles, INDEGSAL and Department of Molecular Biology, University of León, 24071 León, Spain.

E-mail: robles.vanesa@gmail.com


Cryopreservation of Primordial Germ Cells (PGCs) for gene banking in fish is very promising considering the limitations that exist in fish embryo. Via germ line chimerism, these cells allow surrogate production in teleost fish. However, cryopreservation involves a range of extreme factors that might be stressful to cells. Moreover, Reactive Oxygen Species (ROS) produced during cryopreservation can damage DNA. Quantitative PCR was validated to quantify DNA damage mediated directly by reactive oxygen species or by cellular consequences of exposure to ROS. In this study, quantitative PCR (q-PCR) was used for the first time to measure mitochondrial and nuclear DNA damage after PGCs cryopreservation. Our data showed that the smallest number of lesions per 10 Kb was observed in one of the regions of the nuclear genome (2.6). However, nuclear genome was generally more sensitive than mitochondrial genome to damage induced by H2O2 or freezing without cryoprotectants. Using our cryopreservation protocol, the number of DNA lesions per 10 Kb was never higher than 4.68. In summary, q-PCR represents a feasible technique to quantify DNA damage, and this study demonstrates for the first time that it could be successfully applied to analyze PGC protocols.