Document Type thesis Author Name Troccolo, Paul M URN etd-010907-120136 Title Enhanced Animal Cloning: the Effects of Demecolcine on the Anaphase Promoting Complex in Mammalian Development Degree MS Department Biology & Biotechnology Advisors Eric Overstrom, Advisor Samuel Politz, Committee Member David Adams, Committee Member Keywords encleation cloning demecolcine Date of Presentation/Defense 2006-10-11 Availability unrestricted
The efficiency of somatic cell nuclear transfer has been improved slightly with the use of Demecolcine as a chemical enucleant. While the reasons for this improved efficiency remain unclear, it has been hypothesized that the Demecolcine assisted enucleation procedure is less exigent to vital cell processes within the oocyte including the Anaphase-Promoting Complex (APC) dependent ubiquitination of proteins. In order to test the effect of Demecolcine on the APC, the spatial localizations of Apc11, the catalytic core of the complex, and Cdc20, a main activator of the complex, were studied in developing mouse oocytes. In control oocytes, a high concentration of Apc11 protein was observed surrounding the meiotic spindle, but this perispindular localization was not observed in oocytes treated with Demecolcine. Similarly, oocytes stained for Cdc20 also demonstrated cytoplasmic localization in control oocytes with a variation consistent with previous studies in total protein at different stages of development. However, in oocytes treated with Demecolcine, this developmental variation was not observed. These data suggest that since both Apc11 and Cdc20 localization are affected by an incubation in Demecolcine, the activity of the APC would also be affected. In order to test this theory, Rec8, a meiotic specific member of the cohesion complex, was localized in developing mouse embryos. Since the destruction of Rec8 is a downstream consequence of the ubiquitination pathway, Rec8 localization serves as an indirect indicator of APC activity. The data indicate Rec8 localization was only subtly influenced by Demecolcine, thus the magnitude of the drug’s effect APC activity remains unclear.
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