| An efficient system for genetic modification and large scale cloning of cattle is of importance for agriculture, biotechnology and human medicine. Two approaches were investigated here to produce a transgenic bovine. First, the use of embryonic stem (ES) cells in production of chimeras and second, the use of nuclear transfer procedures using genetically modified somatic cells as nuclear donors.; Embryonic stem (ES) cells could be useful for generating transgenic cattle or cells for xenotransplantation. The in vitro production of tissue for xenotransplantation requires the development of an unlimited source of pluripotent cells. Furthermore, these cells should be capable of being genetically modified to aid in directing differentiation, reducing graft rejection, improving graft survival and for producing therapeutic proteins in vivo. A novel method was developed, using nuclear transplantation, to produce transgenic ES cells from fetal bovine fibroblasts. These cells, when reintroduced into preimplantation embryos, were able to differentiate into derivatives from the three embryonic germ layers; ectoderm, mesoderm and endoderm in adult animals. Six out of seven calves born were found to be chimeric for at least one tissue (86%). This work demonstrates that somatic cells can be genetically modified and then dedifferentiated by nuclear transfer into ES-like cells, opening the possibility of using them in differentiation studies and human cell therapy.; In order to produce a whole transgenic bovine in one generation, fetal fibroblasts were genetically modified with a marker gene, a clonal line was selected and the cells were fused to enucleated mature oocytes. Out of 130 embryos transferred to 53 recipient cows, seven healthy, identical, transgenic calves were generated. Furthermore, the lifespan of near senescent fibroblasts could be extended by nuclear transfer, as indicated by population doublings in fibroblast lines derived from a 40 day fetal clone. With the ability to extend the lifespan of these primary cultured cells, this system should be very useful for inducing complex genetic modifications in cattle. |
