Doctor of Philosophy (PhD)
Animal Science (Animal, Dairy, and Poultry Sciences)
Investigations into the importance of the nuclear integrity of the donor cell prior to nuclear transfer (NT) are limited. In Experiment 1, the proliferative characteristics, chromosomal stability and level of histone phosphorylation in cell lines established by explants and enzymatic dissociation at different population doublings (PDs) were investigated. The cells divided at a constant rate and cell cycle length increased only at the end of the proliferative stage. The level of aneuploidies was high and remained elevated throughout the study independent of the technique used to establish the primary culture. High levels of multinucleated cells and abnormal spindle configurations were observed after prolonged time in culture. An increase in the level of phosphorylated histones occurred after extended time in culture. In Experiment 2, gene expression patterns of chromatin remodeling proteins and levels of DNA methylation and histone acetylation of cells were analyzed. Dnmt-1, MeCP-2 and HDAC-1 expression decreased shortly after establishment of the primary culture. Methylated DNA patterns changed with time in culture, while acetylated histone levels remained constant. Embryo production, developmental potential and gene expression patterns of pre- and post-hatched embryos generated using donor cells with different levels of Dnmt-1 were examined in Experiment 3. A higher proportion of 8-16 cell embryos developed to the blastocyst stage when cells with low levels of Dnmt-1 were used as donor nuclei. Day 13 NT embryos generated using donor cells with decreased levels of Dnmt-1 and able to reach the 8-16 cell stage produced a larger number of apparently normal developing embryos, larger conceptuses and higher expression of Dnmt-3a than NT embryos reconstructed using cells with elevated levels of Dnmt-1. However, abnormal gene expression of Dnmts, INFτ and MHC-1 were noted in the majority of cloned embryos indicating inefficient nuclear reprogramming and retarded embryo development. In conclusion, it is likely that the chromosomal abnormalities observed in donor cells at late PDs impair early development of cloned embryos; however, a lower Dnmt-1 content at the time of NT may facilitate the demethylation process during the first divisions resulting in higher development rates in those surviving the 8-16 cell stage.
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Giraldo Gomez, Angelica Maria, "Chromosomal stability and epigenetic modifications of fibroblast cells used for nuclear transfer" (2007). LSU Doctoral Dissertations. 1647.