Abstract
The nuclear reprogramming of somatic cell to an embryonic state has several applications, such as basic research in developmental biology, cell therapy in medicine, genetic improvement of livestock animals and species conservation. The two principal techniques used to achieve nuclear reprogramming are Somatic Cell Nuclear Transfer (SCNT) and Induced Pluripotent Stem Cell (iPS). There are many studies reporting the low efficiency of these techniques. Several researchers have shown that epigenetic modifications, such as DNA methylation and Histone methylation and acetylation, are the main barrier to an efficient reprogramming. Recently, it was described a new kind of epigenetic modification, 5-hydroxymethylcytosine (5hmC), this modification is related to active demethylation of DNA, and it offers a new approach to understand the the mechanisms underlying the nuclear reprograming process. Thus, the aim of this project is investigate the influence of 5hmC on nuclear reprogramming, as well as use new strategies to improve SCNT and iPS efficiency. For this reason, we will analyze the epigenetic patterns (principal markers: 5mC, 5hmC, H3K9ac and H3K9me2) of cells with different states of cellular differentiation (i.e mesenchymal stem cells vs fibroblasts) and we will use them as nuclear donors in SCNT. Moreover, to further investigate the DNA oxidation through 5hmC mechanism. To this end, we will treat different cell lineages with a chromatin-modifying agent (BIX-01294) to use them in nuclear transfer and induction pluripotency experiments. Therefore, we will test whether or not if the levels of oxidised DNA are correlated to the ability of the cells to undergo a complete nuclear reprograming, by global DNA demethylation.
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