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Quantification of DNA damage by q‐ PCR in cryopreserved zebrafish P rimordial G erm C ells
Author(s) -
Riesco M. F.,
Robles V.
Publication year - 2012
Publication title -
journal of applied ichthyology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.392
H-Index - 62
eISSN - 1439-0426
pISSN - 0175-8659
DOI - 10.1111/jai.12089
Subject(s) - cryopreservation , biology , cryoprotectant , dna damage , mitochondrial dna , nuclear dna , genome , dna , real time polymerase chain reaction , embryo , gene , microbiology and biotechnology , genetics
Summary C ryopreservation of P rimordial G erm C ells ( PGC s) 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, R eactive O xygen S pecies ( 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 PGC s 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 H 2 O 2 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.