Bovine nuclear transfer embryo development using cells derived from a cloned fetus

Many different cell types have been used to generate nuclear transfer embry os and fetuses. However, little is known about the potential of fibroblasts derived from a nuclear transfer fetus as donor cells for nuclear transfer. The ability of cloned fetuses or animals to be cloned themselves is of gre at interest in determining whether successive generations of clones remain normal or accumulate genetic or phenotypic abnormalities. We generated a bo vine fibroblast cell line from a cloned fetus, that continued to divide bey ond 120 days (94 doublings,18 passages) in continuous culture. As long-term survival of cells in culture is a desirable characteristic for use in tran sgenic cell production, passage 2 and 18 cells were compared as donor cells for nuclear transfer (NT). When cells from passage 2 (2 weeks in culture) and passage 18 (4 months in culture) were used for nuclear transfer, there was no significant difference in development rate to blastocyst (35.3 versu s 44.6%, P = 0.07). A greater proportion of late passage cells were in G0/G 1 whether under serum-fed (64 versus 56%, P < 0.01) or serum-starved (95 ve rsus 88%, P < 0.01) culture conditions. Following embryo transfer, equivale nt day 30 pregnancy rates were observed for each group (P 2:2/19 versus P 1 8: 2/13). A slightly retarded fetus was surgically removed at day 56 and th e remaining three fetuses died in utero by day 60 of gestation. Our results show that fibroblast cells derived from regenerated cloned fetuses are cap able of both in vitro and in vivo development. The longevity of this regene rated cell line would allow more time for genetic manipulations and then to identify stable transfected cells prior to their use as NT donor cells. Al though no live fetuses were produced in this study the results provide enco uraging data to show that a cloned fetus can itself be recloned to produce another identical cloned fetus. Further studies on this and other recloned fetuses are necessary to determine whether the failure to produce live offs pring was a result of inadequate sample size or due to the cell type select ed. (C) 2001 Elsevier Science B.V. All rights reserved.