Nuclear Transfer Using B6CBAF1 Cytoplasts
In five replicate experiments using B6CBAF1 oocytes, activated and MII cytoplasts were compared in nuclear transfer experiments with HM-1 ES cell nuclei. Again, in each experiment, a sample of oocytes was also partheno-genetically activated with ethanol and cultured to the blastocyst stage. As with B6D2F1, high rates of parthenoge-netic development were observed, with 92% and 84% of activated oocytes cleaving and forming morulae/blastocysts, respectively (n = 93).
A total of 1154 oocytes were activated and treated with demecolcine, of which 673 (58%) were selected 90 min pa for mechanical removal of PBs on the basis of having either single long flat or twin PB phenotypes. Of these oocytes, 360 (31% of the total activated and treated with demecol-cine) survived PB removal and, therefore, were injected. As for B6D2F1, those oocytes possessing a long flat PB tended to survive mechanical PB removal better than those with two PBs (data not shown). Also, generally all B6CBAF1 MII oocytes survived mechanical enucleation. Of 203 enucleated MII oocytes, 193 (95%) survived injection and were activated with SrCl2. In vitro development of cloned embryos from B6CBAF1 MII cytoplasts was comparable to that previously described for B6D2F1 and also resulted in live offspring (Table 6). Activated B6CBAF1 cytoplasts yielded cleavage and morula/blastocyst rates of 36% and 14%, respectively, and unlike previous experiments with B6D2F1 oocytes, the birth and survival of a single live offspring was observed. my canadian pharmacy online