The use of surrogate ooplasm and mtDNA supplementation to enhance nuclear transfer outcome for another species.

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Mitochondria are found in all eukaryotic cells and are the cell?s major generators of ATP through the process of oxidative phosporylation which takes place via the electron transfer chain (ETC). Unlike all other mechanisms in the cell, the ETC is encoded by both the chromosomal and mitochondrial genomes. Normally the mitochondrial genome (mtDNA) is inherited only through the oocyte at fertilisation. However, nuclear transfer (NT), the fusion of a donor cell with an enucleated oocyte violates this pattern and can result in two or more populations of mtDNA being transmitted. Furthermore, NT embryos prematurely express the mtDNA-nuclear encoded replication factors that are translocated to the nucleus unlike their in vitro fertilisation counterparts. The success of NT is low and requires a high number of recipient oocytes. Consequently, for therapeutic cloning, other strategies are required. One approach is the use of surrogate oocytes from another species supplemented with mtDNA matching that of the accompanying donor cell. This would test the hypothesis that successful NT in surrogate oocytes is dependent on the regulation of nucleo-cytoplasmic compatibility. This is because the nucleus may encode proteins for the ETC that are very diverse from those encoded by mtDNA. From our preliminary data, we know that those porcine oocytes not having reached full growth have the capability to replicate mtDNA whilst fully grown oocytes do not. These oocytes not having reached full growth have poor fertilisation outcome but mitochondrial supplementation can enhance this. We would initially supplement both fully grown and non-fully grown oocytes with varying quantities of mitochondria from i) murine, ii) porcine oocytes or iii) a combination of both and then allow these to mature in culture before fertilisation with sperm. We would assess rates of development, and murine mtDNA replication efficiency at each stage of development. Based on these outcomes, we would enucleate supplemented oocytes and perform NT with pluripotent murine embryonic stem cells and adult murine fibroblasts. The resultant embryos would be cultured to various stages of development. We would analyse the ratio of murine and porcine mtDNA using allele-specific PCR and the ability of these embryos to replicate mtDNA through immunocytochemistry and real time RT-PCR. The extent of nucleo-cytoplasmic interaction would be demonstrated through mtDNA-specific polymerase targeted inhibition experiments. Finally, we would generate embryonic stem cells from the resultant blastocysts and determine the extent of murine mtDNA transmission and the pluripotent status of these cells.