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Title: Dynamics of DNA methylation in normal and cloned sheep development
Author: Fairburn, Hannah R.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2002
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The ovine homologues of DNA methyltransferases DNMT1, DNMT3A and DNMT3B, and the methyl-CpG-binding protein MBD2, were cloned fully or partially, using a combination of RT-PCR, cDNA library screening, and 5’ RACE methods. Analysis of predicted protein sequences demonstrated high levels of conservation within and out with functional domains. Attempts to clone an oocyte-specific from of DNMT1, known to exist in the mouse, were unsuccessful. Instead, a form identical to that isolated from adult tissues was detected in oocytes.  These results imply there is little or no oocyte-specific form of Dnmt1 in sheep oocytes, which may be indicative of species-specific differences during early epigenetic reprogramming events. A methyl-sensitive PCR method, optimised for use in single oocytes and embryos, was employed to assay DNA methylation levels of an ovine satellite sequence, and an ovine SINE element. No significant changes were detected in the satellite sequence of in vivo derived embryos between the 8-16 cell and blastocyst stages of development, contrasting with the significant decrease in Satellite DNA methylation levels observed in the mouse. However, comparisons between in vivo, in vitro fertilised (IVF) and SCNT blastocysts demonstrated significant differences, with the IVF and SCNT embryos (both of which undergo equivalent periods of in vivo culture) relatively undermethylated within the satellite and SINE element. In addition, SCNT blastocysts demonstrated increased methylation levels relative to their IVF counterparts within the Satellite sequence, but not the SINE element, suggesting sequence-specific reprogramming errors. In conclusion, there is strong evidence of overall conservation of DNA methylation mechanisms between sheep and mice however, there are subtle differences. In addition, SCNT blastocysts appear to show aberrant reprogramming of methylation patterns when compared to their normal counterparts. This may underlie the low success rates observed in animal SCNT. Future work will build on these results, investigating global methylation levels during preimplantation development of normal and cloned embryos, and also determining the precise roles of ovine DNA methyltransferases in methylation reprogramming.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available