PRODUCTION OF CLONED LAMBS FROM AN ESTABLISHED EMBRYONIC-CELL LINE - A COMPARISON BETWEEN IN-VIVO-MATURED AND IN-VITRO-MATURED CYTOPLASTS

Nuclear transfer procedures were used to determine the in vivo develop mental potential of an ovine embryonic cell line isolated from the inn er cell mass of a Day 8 blastocyst-stage embryo. This cell line posses sed a differentiated epithelial-like cell morphology. In this study, a comparison was made between in vivo- and in vitro-derived oocytes use d as recipient cytoplasts in the nuclear transfer procedure. Cultured cells were induced to quiesce and enter presumptive G0 before being us ed as donor karyoplasts between passages 8 and 16 of culture. After ce ll fusion, reconstructed embryos were cultured for 6 days in vitro in embryo culture medium. Blastocyst-stage embryos were subsequently tran sferred to synchronized recipient ewes (n = 37), and development was a llowed to proceed to term. There was a significant effect of source of recipient cytoplast, with development being consistently greater with in vivo compared to in vitro cytoplasts in terms of, respectively, bl astocysts produced (24.2 +/- 3.8% vs. 17.1 +/- 2.3%; p = 0.1), Day 35 pregnancy rate (40.0% vs. 9.1%; p < 0.05), and Day 35 embryo survival (19.4% vs. 4.5%; p < 0.05). A high proportion of fetuses died during l ate gestation (5 of 8). The major abnormalities were associated with t he urogenital tract. However, three lambs were delivered alive followi ng cesarean section on Day 147. One lamb, derived from an in vitro-mat ured oocyte, died after 10 min, while the remaining two from in vivo-o vutated oocytes are apparently normal and healthy. DNA microsatellite markers conclusively show that the three lambs are genetically identic al and were derived from the embryonic cell line. In conclusion, some cells from this blastocyst-derived embryonic cell line are totipotent by nuclear transfer and can produce viable offspring.