IMPROVEMENT OF STEM CELL AND NUCLEAR CLONING TECHNOLOGIES IN UNGULATES
Project Number: 1265-31000-092-00
Start Date: Jul 24, 2007
End Date: Jul 23, 2012
1. Improve the efficiency of somatic cell nuclear transfer (NT). Protein expression of the tissues of pre-implantation NT, IVF (in vitro fertilized), and parthenogenic (P) 8-day and 16-day embryos (blastocysts) will be compared to identify techniques or conditions that can be altered to create NT embryos that will develop to term at higher frequency. 2. Establishment and characterization of ungulate embryonic stem cell (ESC) lines. Porcine or bovine epiblast tissue from the preimplantation embryo will be assayed for stem cell pathway gene expression and will be cultured under novel experimental conditions. 3. Establishment and characterization of ungulate somatic stem cell (SSC)lines. Porcine or bovine bone marrow or umbilical cord tissue will be cultured to establish cell lines of cells expressing SSC markers.
Cellular protein expression IVF-, NT-, and P-derived embryos will be compared by 2-D gel electrophoretic analysis of bovine trophectoderm and endoderm cell lines derived from blastocyst stage embryos. A composite 2-D gel and protein data base defining both overall protein expression and differential protein expression (potential reprogramming “markers”) from bovine NT and IVF preimplantation embryo-derived trophectoderm cell lines will be produced. These results will be compared to the protein profiles of in vivo-derived 16-day pre-implantation bovine embryos acquired from superovulated and artificially inseminated (AI) heifers. Tests for real-time assessment of the proper reprogramming of NT embryos will be devised based on detection of reprogramming “markers” by immunocytochemical assay, anti-viral assay, and ELISA. The undefined problems inherent to the establishment of ungulate ESC lines will be investigated by candidate-gene-directed RT-PCR and immunocytochemical analysis of the tissues of early preimplantation porcine embryos, particularly the cells constituting the epiblast (in vivo ESC equivalent). Novel culture methods, condition, and constituents will be tested for the continuous culture of ungulate ESC. The establishment of ungulate SSC lines will be attempted from the culture of bovine or porcine bone marrow or umbilical cord cells. Specific markers for the demonstration of pluripotency of putative SSC populations, i.e., differentiation into multiple cell types of one germ layer or more, will be assayed by immunocytochemistry.