Development of transfection technique of foreign gene using bovine ES-like cells as a vector

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The present was carried out to develop a new system for foreign gene transfer into bovine embryos using bovine embryonic stem (ES) cells or somatic cells as a vector. 1. In order to construct a reporter gene, PGK promoter region was inserted into a cloning site of green fluorescent protein (GFP) gene which show a self fluorescent. 2. Electroporation or lipofection methods were used to transfect GDP gene into bovine somatic cells as mammary cells, ear cells or fibroblast. It was shown that electroporation was effective for mammary cells and lipofection method was effective for bovine adult ear cells and fetal fibroblasts in transfection of GDP gene. 3. Six calves were born from the transfer of the chimeric embryos, two calves died at birth or a day after birth. The microsatellite DNA analysis showed that the cells of liver, roots of hair from calves and placenta proved to be chimeric and derived from ES-like cells and tetraploid embryos. 4. We attempted aggregation of diploid or tetraploid embryos with GFP positive ES cells which were selected for only 8 days with G418. At 15 days, 7 normal embryos were obtained, 1 out of 7 embryos showed GFP expression on main five organs, and 4 embryos were detected strongly GFP expression on more one organ under a stereotypic microscope. These results indicate that transfected ES cells or somatic cells selected in a short term with G418 were able to use enough for the selection of preimplantation transgenic embryos and for aggregation chimera.