Somatic cell nuclear transfer (SCNT) is an inefficient process. Our goal is to improve the development of SCNT embryos by driving fibroblast metabolism toward blastomere metabolism before and after SCNT as a way to improve development of the cloned embryo. Our overarching thesis is: Fibroblasts prior to SCNT or embryos after SCNT, driven pharmacologically toward a blastomere metabolism will make the transition from fibroblast to blastomere metabolism less dramatic and result in improved development of the embryo. Our ideal methodology would be to treat the cells to induce a blastomere-like metabolism beginning after genetic modification, or alternatively the day before SCNT. If it is necessary to begin the treatment during selection for the genetic modification, then we must confirm their ability to be expanded, form colonies and survive cryopreservation.
The Specific Aims will be to: 1) Characterize treatment of the donor cells to be more blastomere- like, 2) Treat the donor cells prior to SCNT, 3) Treat the SCNT embryos only, 4) Treat the donor cells and/or the SCNT embryos, and evaluate development to term. Hypothesis 1: Fibroblast cells can be driven toward a blastomere-like (WE) metabolism. Fibroblast cells will be treated with several compounds/conditions, individually and in combination, that would drive them toward a blastomere or Warburg pathway. Hypothesis 2: Fibroblasts with a blastomere metabolism will result in SCNT embryos that have more of an embryo metabolism than those derived from control fibroblasts, and will have better post-attachment development. The treatment from Hypothesis 1 that results in the most blastomere-like metabolism will be used to treat donor cells to create SCNT embryos. Hypothesis 3: SCNT embryos induced to have a blastomere-like metabolism will develop better than control embryos. These experiments will determine if treatment of the SCNT embryo will induce a more blastomere-like metabolism and develop at a higher rate to post-attachment stages. Hypothesis 4: Fibroblasts and SCNT embryos driven to a blastomere-like metabolism will result in SCNT embryos that have more developmental competence than controls. This experiment will combine the efforts of treating the donor cells to become more blastomere-like with the treatment that enhances development of SCNT-treated embryos. In this experiment donor cells will be treated or used as controls (based on Hypothesis 2 results). In addition, SCNT embryos will be treated or used as controls (based on Hypothesis 3 results). Thus we propose a 2X2 factorial treatment structure. Embryos will be cultured to the blastocyst stage and evaluated or transferred. Endpoints will be identical to Hypothesis 3. Overall we expect that adjusting the metabolism of the donor cell prior to SCNT and/or the embryo after SCNT will result in improved development between the blastocyst stage and day 35 of development.
This project seeks to improve the efficiency of somatic cell nuclear transfer in the pig. Swine are important in biomedical research as after genetic engineering they are excellent models for diseases. Genetic engineering of swine has made and will make the pig an even more useful model of human disease. Genetic engineering in somatic cells followed by SCNT will probably remain the method of choice for making these more elegant modifications that are required.
