The long term goal of this research project is to develop an efficient resource for producing transgenic pigs by understanding the basic cellular and molecular biology of the system. This resource will be very valuable for research where the pig is an appropriate model system. To work toward that goal, our earlier funding demonstrated the utility of the tool that we built to perform differential methylation hybridization, whereas here we want to further validate this tool so that it is available as a resource to the NIH community. We propose the following. 1st, we hope to provide a better understanding of early embryo development and the process of differentiation so as to provide clues to methods that may alleviate the losses that occur during the first month of gestation. 2nd, a better understanding of the molecular events that occur to donor cells as well as to early embryos will help to improve the methods of nuclear transfer, the cloning of pig embryos, and the production of genetically modified pigs. This is critical as nuclear transfer is the only technology available for knocking-out or -in a gene. 3rd, since the topic of this proposal deals with DNA methylation, a better understanding of DNA methylation changes during development will provide clues as to the process of imprinting in Sus scrofa. To that end we propose to bring to bear the most relevant technologies into a systems or integrated approach, i.e. 1. the most germane transcriptional analysis platform available (because it was constructed based on the messages that are most abundant in the relevant tissues), 2. a pig genome-based platform designed to discover genomic regions that are differentially methylated, 3. a system of in vitro embryo production, nuclear transfer/cloning and transgenic pig production that is highly efficient at producing embryos and offspring.
The Specific Aims represent hypothesis-driven research that will address three important questions that remain regarding differential DMA methylation, transcriptional profiling and the process of cloning.
The first aim will be to correlate the transcriptional profile with the pattern of DNA methylation in normal and cloned blastocyst stage embryos.
The second aim i s to perform a similar experiment at later stages of development, i.e. days 10 through 14, and to separate the inner cell mass from the trophectoderm.
The third aim i s to determine the methylation profile and transcriptional profile of two different types of stem cells as compared to fetal-derived fibroblast-like cells.
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