The long range aim of this phase of our program is to produce transgenic animals with site specific targeted genetic modifications. Two of the problems associated with the production of transgenic animals is production of mosaic embryos and lack of control over the site of integration.
The specific aim of this proposal is to develop a method to create chimeras such that the cells of interest are directed to contribute to the germ cells of the developing embryo.
This specific aim will be achieved by taking advantage of two developmental characteristics: 1) in mouse chimeras, tetraploid cells do not contribute to a fetus, & 2) smaller more advanced cells in the center of an asynchronous pre-compact chimeric embryo tend to contribute to the inner cell mass of the developing embryo. Standard methods of producing transgenic animals result in a large percentage of embryos that are mosaic, i.e. some cells are transgenic while others are not. This will result in either a transgenic animal that is a mosaic, or alternatively, the transgenic cells ma be selected against and the resulting embryo completely derived from the non-transgenic cells. The rate of mosaicism is higher in domestic animals than in mice. We propose to test this thesis: Chimeric pig embryos composed of tetraploid and diploid cells can result in a single synchronous embryo and give rise to a conceptus that has a diploid fetus and a chimeric (diploid and tetraploid) placenta. In the initial experiment blastomeres from these 8- cell stage embryos will be chimerized with tetraploid blastomeres peri- compaction. Genetically characterized NIH miniature swine boars will be used as an aid in identifying the chimeric cells. Development and relative contribution from each cell type will be at compaction, day 30 and at term. In the second half of the experiments transgenic embryos will be created. Mosaicism will be confirmed at the 8-cell stage by including in the construct a coding region for the green fluorescent protein. These presumptive transgenic cells will be disaggregated and placed with blastomeres of previously created tetraploid 4-cell stage embryos. In both cases it is expected that the tetraploid cells will only contribute to the placental tissues, while the normal or transgenic cells will comprise the fetus. A similar experiment is designed to determine if inner cell mass cells will have the same potential when chimerized with either a tetraploid blastocyst or a tetraploid peri-compaction embryo. The latter study will lay the ground work for the application of embryonic stem cell technology when it becomes available.
