The long-range goals of the proposed experiments are to understand the basic biology behind nuclear remodeling and reprogramming that occurs when nuclei are transferred to the cytoplasm of oocytes. This understanding will permit development of a system for creating transgenic pigs via nuclear transfer. Interestingly, many investigators, including the applicant (see the preliminary results), have observed that some cells are more effective than others in promoting development to the blastocyst stage, and to the production of live offspring. While differences can be a result of the tissue from which the cell line was established, how long it was cultured in vitro, as well as the in vitro conditions, in our situation the different sublines were all derived from the same fetus. It is clear that in vitro conditions can result in changes in gene expression and DNA methylation status, but it is not clear why those changes occur. Since DNA methylation status is not reprogramable, and since high levels of methylation are generally associated with the repression of transcription, there is considerable justification for evaluating the DNA methylation status in donor cells as well as during embryogenesis.
The Aims are to: 1. Determine the methylation status of at least two cell lines that have high and low developmental potential sub-lines after nuclear transfer, 2 identify specific DNA sequences upon which differential methylation occurs during preimplantation embryo development in the pig, 3. define culture conditions that are more and less likely to result in changes in DNA methylation for cells cultured in vitro as well as for early embryos, and 4. predict and test which cell lines (based upon methylation status of specific sequences) will result in development after nuclear transfer and those that will not work well. Nuclear transfer is a procedure by which not only genetically identical individuals can be created, but which will aid in making specific genetic modifications prior to creating the animal. The applications of these techniques for nuclear transfer will be in agricultural, biomedical, and basic research. Here, the investigators will evaluate a few specific parameters of what makes nuclear transfer successful or unsuccessful.
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