69 APPLICATION OF BOVINE AMNIOTIC CELLS FOR PRENATAL GENETIC DIAGNOSIS AND NUCLEAR TRANSFER

Abstract

Amniotic fluid includes many cells derived from the fetus called 'amniotic cells'. Although these amniotic cells may have much potential as a reproduction or breeding source of the animals, there has been limited study of the potential applications of these cells. We examined the potential of bovine amniotic cells for biotechnological use. Bovine amniotic cells separated from amniotic fluid obtained from a slaughterhouse were prepared for use in all experiments. First, to examine the culture condition of amniotic cells, the cells were cultured in various culture media. Cytologic normality of the cultured cells was analyzed by chromosomal examination and Papanicolaou examination. Second, to examine the potential of cultured amniotic cells for prenatal genetic diagnosis, the cells were used for sexing by PCR. The coincidence of the results with the gender of the fetus from which the cells were derived was examined. Third, we used the cultured cells as donor cells for nuclear transfer, and examined the developmental ability of reconstructed embryos. The normality of the blastocysts derived from the reconstructed embryos was examined by chromosomal examination and transplantation to the recipient heifer. Bovine amniotic cells were cultured successfully in Amnio-max C-100^® (GIBCO, Grand Island, NY, USA), which is marketed as culture medium for human amniotic cells. In all cases, the sex of cultured amniotic cells analyzed by PCR was coincident with that of the fetus from which the amniotic cells were derived. The frequencies of cleavage and development to the blastocyst stage of embryos reconstructed from amniotic cells were the same as those of fetal fibroblasts. There were no differences in the normality of chromosomal number between blastocysts derived from amniotic cells and fetal fibroblasts. A blastocyst derived from amniotic cells developed into a fetus after transplantation. DNA microsatellite analysis of the fetus at Day 64 was coincident with that of the fetus from which the amniotic cells were derived. These results indicate that bovine amniotic cells can be successfully cultured in vitro, and the cultured cells precisely reflect the genetic information of the fetus from which the cells were derived. The cultured cells also have developmental ability as donor cells for nuclear transfer. Amniotic cells may have the potential for effective reproduction and breeding using genetic and biotechnological sources.