Ovine primary fetal fibroblasts were cotransfected with a neomycin resistance marker gene (neo) and a human coagulation factor IX genomic construct designed for expression of the encoded protein in sheep milk. Two cloned transfectants and a population of neomycin (G418)-resistant cells were used as donors for nuclear transfer to enucleated oocytes. Six transgenic lambs were liveborn: Three produced from cloned cells contained factor IX and neo transgenes, whereas three produced from the uncloned population contained the marker gene only. Somatic cells can therefore be subjected to genetic manipulation in vitro and produce viable animals by nuclear transfer. Production of transgenic sheep by nuclear transfer requires fewer than half the animals needed for pronuclear microinjection.
Nuclear transfer offers a cell-based route for producing precise genetic modifications in a range of animal species. Using sheep, we report reproducible targeted gene deletion at two independent loci in fetal fibro-blasts. Vital regions were deleted from the alpha(1,3)galactosyl transferase (GGTA1) gene, which may account for the hyperacute rejection of xenografted organs, and from the prion protein (PrP) gene, which is directly associated with spongiform encephalopathies in humans and animals. Reconstructed embryos were prepared using cultures of targeted or nontargeted donor cells. Eight pregnancies were maintained to term and four PrP-/+ lambs were born. Although three of these perished soon after birth, one survived for 12 days. These data show that lambs carrying targeted gene deletions can be generated by nuclear transfer.
To clone a pig from somatic cells, we first validated an electrical activation method for use on ovulated oocytes. We then evaluated delayed versus simultaneous activation (DA vs. SA) strategies, the use of 2 nuclear donor cells, and the use of cytoskeletal inhibitors during nuclear transfer. Using enucleated ovulated oocytes as cytoplasts for fetal fibroblast nuclei and transferring cloned embryos into a recipient within 2 h of activation, a 2-h delay between electrical fusion and activation yielded blastocysts more reliably and with a higher nuclear count than did SA. Comparable rates of development using DA were obtained following culture of embryos cloned from ovulated or in vitro-matured cytoplasts and fibroblast or cumulus nuclei. Treatment of cloned embryos with cytochalasin B (CB) postfusion and for 6 h after DA had no impact on blastocyst development as compared with CB treatment postfusion only. Inclusion of a microtubule inhibitor such as nocodozole with CB before and after DA improved nuclear retention and favored the formation of single pronuclei in experiments using a membrane dye to reliably monitor fusion. However, no improvement in blastocyst development was observed. Using fetal fibroblasts as nuclear donor cells, a live cloned piglet was produced in a pregnancy that was maintained by cotransfer of parthenogenetic embryos.
A crossbreeding trial extending over three generations was used to investigate the genetic components contributing to the prolificacy of the Meishan breed in comparison with the Large White breed. Information on the number of teats and on body weight and litter size in the first two parities was recorded on purebred Meishan and Large White females and on reciprocal F1 and backcross females. Ovulation rate was also recorded for all litters, allowing the estimation of per litter prenatal survival. Crossbreeding parameters for direct, maternal and grandmaternal effects were estimated using restricted maximum likelihood analysis. There was a consistent advantage of three to four piglets born alive to the Meishan female compared with the Large White female. This was controlled by the maternal genotype, with no effect of the genotype of the litter itself. Both additive and heterosis effects were important, the contribution of additive maternal effects to the breed difference being similar across parities (4·0 (s.e. 1·1) and 4·2(s.e. 1·1), in the two parities respectively) and the maternal heterosis increasing slightly across parities (2·2 (s.e. 0·8) and 2·9 (s.e. 0·8), in the two parities respectively). The number born alive to F 1 females was similar to, or greater than, the number born alive to Meishan females. Ovulation rate was significantly higher in Meishan than in Large White females and this was controlled by additive gene effects which had a similar effect across parities, the weighted average of their contribution to the breed difference being 5·7 (s.e. 0·8) ova. Differences between the breeds in prenatal survival were small, although there was significant maternal heterosis, however, the maternal additive effect became significant after the inclusion of ovulation rate as a covariate. After adjustment for ovulation rate, the weighted average estimates across parity of the maternal additive contribution to the breed difference and the maternal heterosis for the proportional prenatal survival loere 0-14 (s.e. 0·05) and 0·13 (s.e. 0·03), respectively. This suggests that a combination of a high ovulation rate and especially a high level of prenatal survival for that ovulation rate led to the prolificacy observed in this sample of Meishan pigs. The inclusion of ovulation rate as a covariate in the analysis of number born alive confirms this view, as the maternal additive effect on litter size was only reduced by about one third and the heterosis effect was largely unchanged. Both numbers stillborn and mummified were increased in litters born to Meishan sows due to maternal additive effects, but the effects seemed largely a consequence of the increased ovulation rate as they became non-significant after its inclusion as a covariate in the model.
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