Studies on the effect of cryopreservation on gene expression in zebrafish blastomeres
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AbstractCryopreservation is now a common practice in the fields of aquaculture, conservation and biomedicine. The success of cryopreservation is usually analysed in terms of cell survival, although there are other potential adverse effects that don’t necessarily result in cell death. These include DNA damage, which could result in altered gene expression. The aim of this study is to discover if cryopreservation has an impact on gene expression using zebrafish (Danio rerio) as the model organism. As the whole embryo cannot yet be successfully cryopreserved, isolated blastomeres from the embryos were used to investigate the impact of cryo-treatment on gene expression. This study sets out firstly to determine an optimum cryopreservation protocol for 50% epiboly blastomeres (Epiboly displaces the blastoderm margin to 50% of the distance between the animal and vegetal pole). Blastomeres had the highest survival level (70.2 ± 3.2%) when a mixture of 1.5 M dimethyl sulfoxide (DMSO) and 0.1 M sucrose were used as cryoprotectants. As quantitative analysis of gene expression using real-time PCR requires the use of a housekeeping gene as an internal control to normalize date, the second study aimed to identify and validate housekeeping genes for use in cryopreservation studies of zebrafish blastomeres. Seven potential housekeeping genes were analysed across a range of embryo stages and isolated blastomeres using the GeNorm and NormFinder software packages. Results indicated β-actin and EF1α housekeeping genes to be the most appropriate for cryopreservation studies on zebrafish embryos and blastomeres, and these housekeeping genes were used in the third study, the effect of cryopreservation on Pax gene expression. The results indicated that the trends (profile changes) in expression of Pax2a and Pax5 occurred to a lesser extent in frozen-thawed blastomeres than in fresh blastomeres whilst the opposite was true for Pax8. The trends in expression of Pax2b were delayed in frozen-thawed blastomeres compared to fresh blastomeres. Cryopreservation can therefore disrupt normal gene expression patterns in zebrafish embryos which could have a detrimental effect on embryo development. This is the first study on the stability of housekeeping and transcription factor genes in chilled and cryopreserved embryonic cells of the zebrafish. This work will significantly enhance future studies investigating the impact of cryopreservation on gene expression in zebrafish embryos.
PublisherUniversity of Bedfordshire
TypeThesis or dissertation
DescriptionA thesis submitted to the University of Bedfordshire in accordance with the requirements for the degree of Doctor of Philosophy
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